Integrated target waveguide devices and systems for optical coupling

ABSTRACT

Integrated target waveguide devices and optical analytical systems comprising such devices are provided. The target devices include an optical coupler that is optically coupled to an integrated waveguide and that is configured to receive optical input from an optical source through free space, particularly through a low numerical aperture interface. The devices and systems are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices provide for the efficient and reliable coupling of optical excitation energy from an optical source to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination. The devices and systems are well suited for miniaturization and high throughput.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/515,336, filed on Jul. 18, 2019, which is a continuation of U.S.patent application Ser. No. 15/180,016, filed Jun. 11, 2016, now U.S.Pat. No. 10,365,434, which claims the benefit of U.S. ProvisionalApplication No. 62/175,139, filed on Jun. 12, 2015, the disclosures ofwhich are each incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

As multiplexed optical analytical systems continue to be miniaturized insize, expanded in scale, and increased in power, the need to developimproved systems capable of delivering optical energy to such systemsbecomes more important. For example, highly multiplexed analyticalsystems comprising integrated waveguides for the illumination ofnanoscale samples are described in U.S. Patent Application PublicationNos. 2008/0128627 and 2012/0085894. Further optical systems for theanalysis of nanoscale samples, including the illumination and detectionof such samples, are described in U.S. Patent Application PublicationNos. 2012/0014837, 2012/0021525, and 2012/0019828. Additional nanoscaleillumination systems for highly multiplexed analysis are described inU.S. Patent Application Publication Nos. 2014/0199016 and 2014/0287964.

In conventional optical systems, optical trains are typically employedto direct, focus, filter, split, separate, and detect light to and fromthe sample materials. Such systems typically employ an assortment ofdifferent optical elements to direct, modify, and otherwise manipulatelight entering and leaving a reaction site. Such systems are frequentlycomplex and costly and tend to have significant space requirements. Forexample, typical systems employ mirrors and prisms in directing lightfrom its source to a desired destination. Additionally, such systems caninclude light-splitting optics such as beam-splitting prisms to generatetwo or more beams from a single original beam.

Alternatives to the conventional optical systems have been described, inparticular alternative systems having integrated optical componentsdesigned and fabricated within highly confined environments. Forexample, planar lightwave circuits (PLCs) comprising fiber interfaces,wavelength filters or combiners, phase-delayed optical interferometers,optical isolators, polarization control, and/or taps have been developedfor use in telecommunications applications. In some cases these devicesadditionally include one or more laser sources and one or more opticaldetectors. The devices, which are sometimes also referred to as fiberspacing concentrators (FSCs), use integrated optical waveguides to routephotons through an optical circuit, in much the same way as electronsare routed through an electrical circuit. They are fabricated usingstandard semiconductor fabrication techniques, and they can accordinglyintegrate both passive components, such as optical filters and fiberpigtail connectors, and active elements, such as optical switches andattenuators, during the fabrication process. As used intelecommunications equipment, they typically serve to couple and/orsplit optical signals from fiber optic cores, for the purpose of, forexample, multiplexing/demultiplexing, optical branching, and/or opticalswitching. The devices thus provide the functionality of a moretraditional optical train, while at the same time being significantlyless expensive, more compact, and more robust.

In the just-described optical systems, an optical source and its targetdevice are typically closely and permanently associated with one anotherwithin the system. For example, PLCs used in telecommunicationsapplications are typically mechanically aligned and bonded to theirlaser light source and to their associated photodetectors during themanufacturing process. Such close and irreversible associations betweenan optical source and its target device are thus not well suited for usein analytical systems having a removable sample holder, where theoptical output from an optical source, such as a traditional opticaltrain, is normally coupled to the target sample holder through freespace. In systems optically coupled through free space, the opticalsignal from an optical source needs to be aligned with a target deviceeach time the target device is replaced, and the alignment can even needto be monitored and maintained during the course of an analysis, due tomechanical, thermal, and other interfering factors associated with theoptical system. In addition, the integrated optical circuits typicallyused in telecommunications applications are not designed to carry theintensity of optical energy necessary to analyze the large numbers ofnanoscale samples present in the highly-multiplexed analytical systemsdescribed above, nor are they designed for use with optical sourceshaving wavelengths suitable for use in optical systems with standardbiological reagents.

Another consideration in the design of an optical analytical system isthe method of coupling of light from the optical source into the targetdevice. For example, where a target device comprises an integratedoptical waveguide for routing the optical energy through the device,launching of the optical energy into the waveguide can be unreliable andinefficient. Various optical couplers have been described to achievethis purpose, including the use of direct “endfire” coupling into apolished end of the waveguide, the use of a prism coupler to directlight into the waveguide, and the use of a grating coupler to directlight into the waveguide. Depending on the implementation, however, eachapproach has limitations with respect to efficiency, reliability,applicability, cost, and the like.

There is thus a continuing need to improve the performance andproperties of integrated optical waveguide devices, particularly thosethat are reversibly coupled to external light sources. There is also aneed to improve the performance and properties of optical analyticalsystems containing such integrated waveguide devices.

SUMMARY OF THE INVENTION

The present disclosure addresses these and other needs by providing inone aspect an integrated target waveguide device comprising an opticalcoupler and an integrated waveguide optically coupled to the opticalcoupler. In this device, the optical coupler is a low numerical aperturecoupler and is at least 100 μm² in size.

In another aspect, the disclosure provides an integrated targetwaveguide device comprising an optical coupler, an integrated waveguideoptically coupled to the optical coupler, and at least one alignmentfeature. In this device, the optical coupler is also a low numericalaperture coupler and is also at least 100 μm² in size.

In yet another aspect, the disclosure provides an optical analyticalsystem comprising an optical source and any of the integrated targetwaveguide devices disclosed herein. In this system, the optical sourceis optically coupled to the optical coupler of the target waveguidedevice through free space at a distance of at least 1 mm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate differences between coupling from an opticalsource with high numerical aperture, such as a fiber tip, and couplingfrom an optical source with low numerical aperture through free space toa target waveguide device.

FIG. 2A shows a plot of the intensity of a Gaussian beam as a functionof distance from the beam axis. FIG. 2B illustrates the shape of adivergent Gaussian beam of radius w(z).

FIG. 3A shows the basic design features and structure of a standardgrating coupler, and FIG. 3B shows the same for a blazed gratingcoupler. FIGS. 3C to 3L show alternative optical grating coupler designsand structural features.

FIG. 4A shows the input coupling region of an exemplary target waveguidedevice with active waveguide alignment features. FIG. 4B shows in closerdetail the two types of input couplers used in the waveguide device ofFIG. 4A. FIG. 4C shows another exemplary input coupling region thatincludes both waveguide alignment features and patterned regionalignment features. FIG. 4D shows the top view of an exemplaryintegrated target waveguide device, including the input coupling region,routing paths, fanout regions, and the arrayed nanoscale sample region.FIG. 4E shows an exemplary optical analytical system, including anoptical source comprising lasers, a beam power controller, and a “lightbrush” to direct the optical input to the integrated target waveguidedevice. Also shown is an alignment camera. FIG. 4F shows the degrees offreedom to be controlled during the alignment of an optical source and atarget device. The motions are designated along track (AT), cross track(CT), pitch, yaw, and roll (or pattern rotation). Not shown is movementin the up-down dimension.

FIGS. 5A-5D illustrate exemplary grating couplers. FIG. 5A shows a basicgrating coupler. FIG. 5B shows a structure that includes an opticalreflective layer directly below the coupler. FIG. 5C shows a structurewith a heat spreading layer directly below the coupler. FIG. 5D shows astructure with both a reflective layer and a heat spreading layer belowthe coupler.

FIG. 6 illustrates “hotspots” created by a multimode coupler.

FIG. 7 shows the effect of linear polarized excitation light on targetsat different locations in a nanowell/ZMW.

FIG. 8 shows the effect of circular polarized excitation light ontargets at different locations in a nanowell/ZMW

FIG. 9 shows the effect of excitation with different TE modes on targetsat different locations in a nanowell/ZMW.

FIG. 10 shows the pattern of TE, TM, and TEM modes in a rectangularwaveguide

FIGS. 11A-11B illustrate single-look and multi-look coupling withgrating-coupled waveguide devices (A) and endfire-coupled waveguidedevices (B).

FIGS. 12A-12C illustrate single-look (A) and multi-look (B and C)devices configured for illumination by three separate input opticalbeams. The devices include input grating couplers (A and B) or endfirecouplers (C).

FIG. 13 illustrates the use of thermal Mach-Zehnder switches to controlmulti-look illumination in an endfire-coupled target device.

FIG. 14A shows an instrument-level implementation of apolarization-based 2-look system. FIG. 14B shows a device-levelimplementation of a polarization-based 2-look system.

FIG. 15 illustrates the use of an arrayed waveguide grating (AWG) totune excitation wavelengths for multi-look reactions.

FIG. 16 shows a novel fiber spacing concentrator with active corealignment.

FIG. 17A illustrates a 2-dimensional low-NA grating coupler model. FIG.17B illustrates the modeled optical energy coupled through the deviceinto an integrated waveguide, where the optical energy is directed fromthe middle of the device towards the left side of the device.

FIG. 18 provides a comparison of coupling efficiencies for variousbinary grating coupler designs.

FIG. 19 provides a comparison of coupling efficiencies for variousbinary grating coupler designs with different numerical aperture values.

FIG. 20 illustrates fiber-to-grating alignment tolerances at variousnumerical aperture values.

FIGS. 21A-21D illustrate the impact of grating period (A), buried oxidecladding thickness (B), duty cycle (C), and etch depth (D) on couplingefficiency at various numerical aperture values.

FIG. 22 summarizes the simulated efficiencies of exemplary couplersdesigned and simulated using the parameters shown.

FIG. 23A shows the cross section of an exemplary waveguide of theinstant target devices, and FIG. 23B shows the electric field intensitythrough the center of the waveguide.

FIG. 24 shows mode profiles for prototype coupled waveguide devices.

FIG. 25 illustrates the impact of y misalignment on the efficiency ofcoupling.

FIG. 26 illustrates the relationship between the prism refractive indexand the input incident angle for a prism-coupled device.

FIG. 27 illustrates the relationship between the grating period and theinput incident angle for a grating-coupled device.

FIG. 28 shows the experimental setup used to test the effectiveness of aheat-spreading layer in mitigating laser-induced thermal damage.

FIGS. 29A-29G show the results of testing samples containing aheat-spreading layer.

FIG. 30 shows simulations of optimized waveguide dimensions forsingle-mode operation in two different waveguide cores with 552 nmlight.

FIG. 31 shows the Gaussian profile for a simulated input beam source.

FIGS. 32A-32B illustrate a 2-dimensional grating coupler model for atarget waveguide device and the modeled optical energy coupled throughthe device into an integrated waveguide.

FIG. 33 illustrates effects of wavelength on modeled coupling efficiencyfor a high NA grating coupler design with a titanium dioxide core.

FIGS. 34A-34B illustrate effects of grating coupler period on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 552 nm input source.

FIGS. 35A-35B illustrate effects of grating coupler duty cycle onmodeled coupling efficiency for a high NA grating coupler design with atitanium dioxide core and a 552 nm input source.

FIGS. 36A-36B illustrate effects of grating coupler etch depth onmodeled coupling efficiency for a high NA grating coupler design with atitanium dioxide core and a 552 nm input source.

FIGS. 37A-37B illustrate effects of reflector distance on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 552 nm input source.

FIGS. 38A-38B illustrate effects of top clad thickness on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 552 nm input source.

FIGS. 39A-39B illustrate effects of waveguide core index on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 552 nm input source.

FIG. 40 plots modeled coupling efficiency for a high NA grating couplerdesign with a titanium dioxide core and a 532 nm input source.

FIGS. 41A-41B illustrate effects of grating coupler period on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 532 nm input source.

FIGS. 42A-42B illustrate effects of grating coupler duty cycle onmodeled coupling efficiency for a high NA grating coupler design with atitanium dioxide core and a 532 nm input source.

FIGS. 43A-43B illustrate effects of grating coupler etch depth onmodeled coupling efficiency for a high NA grating coupler design with atitanium dioxide core and a 532 nm input source.

FIGS. 44A-44B illustrate effects of reflector distance on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 532 nm input source.

FIGS. 45A-45B illustrate effects of top clad thickness on modeledcoupling efficiency for a high NA grating coupler design with a titaniumdioxide core and a 532 nm input source.

DETAILED DESCRIPTION OF THE INVENTION Optical Analytical Systems

Multiplexed optical analytical systems are used in a wide variety ofdifferent applications. Such applications can include the analysis ofsingle molecules, and can involve observing, for example, singlebiomolecules in real time as they interact with one another. For ease ofdiscussion, such multiplexed systems are discussed herein in terms of apreferred application: the analysis of nucleic acid sequenceinformation, and particularly, in single-molecule nucleic acid sequenceanalysis. Although described in terms of a particular application,however, it should be appreciated that the devices and systems describedherein are of broader application.

In the context of single-molecule nucleic acid sequencing analyses, asingle immobilized nucleic acid synthesis complex, comprising apolymerase enzyme, a template nucleic acid whose sequence is beingelucidated, and a primer sequence that is complementary to a portion ofthe template sequence, is observed analytically in order to identifyindividual nucleotides as they are incorporated into the extended primersequence. Incorporation is typically monitored by observing an opticallydetectable label on the nucleotide, prior to, during, or following itsincorporation into the extended primer. In some cases, such singlemolecule analyses employ a “one base at a time approach”, whereby asingle type of labeled nucleotide is introduced to and contacted withthe complex at a time. In some cases, unincorporated nucleotides arewashed away from the complex following the reaction, and the labeledincorporated nucleotides are detected as a part of the immobilizedcomplex. In other cases, it is possible to monitor the incorporation ofnucleotides in real time without washing away unincorporatednucleotides.

In order to obtain the volumes of sequence information that can bedesired for the widespread application of genetic sequencing, e.g., inresearch and diagnostics, higher throughput systems are desired. By wayof example, in order to enhance the sequencing throughput of the system,multiple complexes are typically monitored, where each complex sequencesa separate DNA template. In the case of genomic sequencing or sequencingof other large DNA components, these templates typically compriseoverlapping fragments of genomic DNA. By sequencing each fragment, acontiguous sequence can thus be assembled using the overlapping sequencedata from the separate fragments.

A single template/DNA polymerase-primer complex of such a sequencingsystem can be provided, typically immobilized, within a nanoscale,optically-confined region on or near the surface of a transparentsubstrate, optical waveguide, or the like. Such an approach is describedin U.S. Pat. No. 7,056,661, which is incorporated by reference herein inits entirety. These optically-confined regions are preferably fabricatedas nanoscale sample wells, also known as nanoscale reaction wells,nanowells, or zero mode waveguides (ZMWs), in large arrays on a suitablesubstrate in order to achieve the scale necessary for genomic or otherlarge-scale DNA sequencing approaches. Such arrays preferably alsoinclude an associated optical source or sources, to provide excitationenergy, an associated emission detector or detectors, to collect opticalenergy emitted from the samples, and associated electronics. Together,the components thus comprise a fully operational optical analyticaldevice or system. Examples of analytical devices and systems useful insingle-molecule nucleic acid sequence analysis include those describedin U.S. Pat. Nos. 6,917,726, 7,170,050, and 7,935,310; U.S. PatentApplication Publication Nos. 2012/0014837, 2012/0019828, and2012/0021525; and U.S. patent application Ser. No. 13/920,037, which areeach incorporated by reference herein in their entireties.

In embodiments, the instant optical analytical systems comprise anoptical source that is coupled to a target device, typically anintegrated target waveguide device. As will be described in more detailbelow, the optical source and the associated target device areconfigured for efficient coupling through free space, for example at adistance of at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm,at least 10 mm, at least 20 mm, at least 30 mm, at least 50 mm, or evenat least 100 mm.

As will also be described in more detail below, it can be advantageousin the efficient coupling of optical energy from the optical source tothe target device for the optical devices to be configured with lownumerical aperture. By “low numerical aperture” it is meant that thenumerical aperture is lower than the numerical aperture of near-fieldcoupled optical devices. Specifically, it is meant that the numericalaperture is no more than 0.1. Accordingly, in some system embodiments,the optical source and the associated target device have numericalapertures of no more than 0.1, no more than 0.08, no more than 0.05, nomore than 0.03, no more than 0.02, or even no more than 0.01.Furthermore, in some embodiments, the optical source is configured toilluminate a spot on the associated target device with a surface areaper spot of at least 100 μm², at least 144 μm², at least 225 μm², atleast 400 μm², at least 625 μm², at least 900 μm², at least 1600 μm², atleast 2500 μm², at least 4900 μm², at least 10,000 μm², or even higher.In other embodiments, the optical source is configured to illuminate aspot on the associated target device with a surface area per spot of atmost 250,000 μm², at most 62,500 μm², at most 22,500 μm², at most 10,000μm², at most 6400 μm², at most 3600 μm², or at most 2500 μm². In stillother embodiments, the optical source is configured to illuminate a spoton the associated target device with a surface area per spot of from 100μm² to 250,000 μm², from 225 μm² to 62,500 μm², from 400 μm² to 22,500μm², from 625 μm² to 10,000 μm², from 900 μm² to 6400 μm², or even from1600 μm² to 3600 μm².

In some system embodiments, the optical source is configured toilluminate a spot on the associated target device with a power of atleast 1 mW, at least 2 mW, at least 3 mW, at least 5 mW, at least 10 mW,at least 20 mW, at least 30 mW, at least 50 mW, or at least 100 mW perspot.

In some system embodiments, the optical source emits a plurality oflight beams. The separate light beams are preferably arranged toilluminate a corresponding plurality of optical input couplers on theassociated target device. Separating the optical energy into multiplebeams can be advantageous in decreasing the input energy per beam andthus decreasing the requirement to dissipate heat energy on the targetdevice. In some embodiments, the optical source emits at least fourlight beams. In specific embodiments, the optical source emits at leasteight light beams or even at least twelve light beams.

In some embodiments, the optical source emits at least one sampleexcitation beam and at least one alignment beam. As will be described inmore detail below, the sample excitation beam is directed through freespace to an input coupler on the target waveguide device and from thereis directed—typically through an array of integrated waveguides—tonanoscale sample wells arrayed on the device. The alignment beam isdirected through free space to an alignment feature on the targetwaveguide device and serves to align the target device and the opticalsource or to maintain such alignment, as will be described in furtherdetail below. In specific embodiments, the alignment beam is of a loweroutput power than the sample excitation beam. In some embodiments, thealignment beam has no more than 10% of the output power of the sampleexcitation beam. More specifically, the alignment beam has no more than5% of the output power of the sample excitation beam or even no morethan 1% of the output power of the sample excitation beam.

Accordingly, in some system embodiments, the target device comprises analignment feature, and the optical system further comprises an alignmentdetector. The combination of an alignment feature on the target deviceand an alignment light beam and alignment detector within the system isparticularly useful in systems where the target device is designed to beremovable. In such a system, when a new target device is installed intothe system, the alignment feature or features on the target device canbe used by the alignment detector to adjust the position of the targetdevice relative to other components of the system, particularly withrespect to the optical source, and can thus optimize the coupling ofoptical energy from the optical source to the target device.

For example, in systems where the optical source emits multiple opticalbeams, such as in some of the integrated optical delivery devicesdescribed in co-owned U.S. Patent Application No. 62/133,965, filed onMar. 16, 2015, and U.S. patent application Ser. No. 15/072,146, filed onMar. 16, 2016, the disclosures of which are incorporated by referenceherein in their entireties, it can be difficult to achieve optimalalignment of the multiple beams with the multiple input couplers of atarget device and to maintain that alignment during the course of ameasurement. The alignment beams and associated alignment features ofthe instant systems overcome those difficulties both by facilitating theinitial alignment of the optical source and the target device within theoptical system and by maintaining that alignment during the course of ananalytical assay.

In particular, the process of aligning an optical source with the targetdevice can include a coarse alignment process, a fine alignment process,or both coarse and fine alignment processes. During the alignmentprocess, the target waveguide device itself can be moved relative to theoptical source, the optical source can be moved relative to the targetwaveguide device, or both devices can be moved relative to one another.In preferred system embodiments, the alignment detector provides for thedynamic alignment of the integrated target waveguide device and theoptical source, such that alignment between the components is maintainedduring an assay. In some system embodiments, the alignment detector is acamera.

As was described in U.S. Patent Application Nos. 62/133,965 and15/072,146, the optical source of the instant systems can provide amodulated optical signal. In specific embodiments, the modulated opticalsignal can be amplitude modulated, phase modulated, frequency modulated,or a combination of such modulations.

In some system embodiments, the optical output waveguide of the opticaldelivery devices comprises a power modulator, such as a variable opticalattenuator or even an integrated optical Mach-Zehnder interferometer.

In some system embodiments, the optical delivery device comprises one ormore splitting elements optically coupled to the optical input of thedevice through an optical input waveguide. The splitting element orelements is optically coupled to a plurality of optical outputs througha plurality of optical output waveguides. In some embodiments, thesplitting element or elements is optically coupled to a tap waveguide,and the optical delivery device can further comprise a power monitoroptically coupled to the tap waveguide. In specific embodiments, thepower monitor can control the power output transmitted by an opticaloutput of the device. In specific embodiments, the power monitor can bean integrated detector or a camera. In some embodiments, the splittingelement or elements is optically coupled to a tap waveguide, and the tapwaveguide is optically coupled to an output grating or a region of thincladding.

In some system embodiments, the optical delivery device comprises anamplitude modulator optically coupled to an optical output of the devicethrough an optical output waveguide. The amplitude modulator can, inspecific embodiments, be a variable optical attenuator.

In some system embodiments, the optical delivery device can comprise amode stripper optically coupled to an optical output of the devicethrough an optical output waveguide. In embodiments, the opticaldelivery device can be configured for optical output of visible light,for example visible light having a wavelength in the range from about500 nm to about 600 nm.

In certain embodiments, the optical source of the instant opticalsystems is one or more lasers, including vertical-cavitysurface-emitting lasers, one or more light-emitting diodes, or one ormore other comparable optical devices. In specific embodiments, theoptical source is one or more lasers.

As already noted, in the analysis of genomic sequence information, itcan be advantageous for the target devices of the instant opticalanalytical systems to include arrays with large numbers of nanoscalesample wells. In order to achieve such scale, the arrays can befabricated at ultra-high density, providing anywhere from 1000 nanowellsper cm², to 10,000,000 nanowells per cm², or even higher density. Thus,at any given time, it can be desirable to analyze the reactionsoccurring in 100, 1000, 3000, 5000, 10,000, 20,000, 50,000, 100,000, 1Million, 5 Million, 10 Million, or even more nanowells or other sampleregions within a single analytical system, and preferably on a singlesuitable substrate.

In order to achieve the ultra-high density of nanowells necessary forsuch arrays, the dimensions of each nanowell must be relatively small.For example, the length and width of each nanowell is typically in therange of from 50 nm to 600 nm, ideally in the range of from 100 nm to300 nm. It should be understood that smaller dimensions allow the use ofsmaller volumes of reagents and can, in some cases, help to minimizebackground signals from reagents outside the reaction zone and/oroutside the illumination volume. Accordingly, in some embodiments, thedepth of the nanowell can be in the range of 50 nm to 600 nm, moreideally in the range of 100 nm to 500 nm, or even more ideally in therange of 150 to 300 nm.

It should also be understood that the shape of a nanowell will be chosenaccording to the desired properties and methods of fabrication. Forexample, the shape of the nanowell can be circular, elliptical, square,rectangular, or any other desired shape. Furthermore, the walls of thenanowell can be fabricated to be vertical, or the walls of the nanowellcan be fabricated to slope inward or outward if so desired. In the caseof a circular nanowell, an inward or outward slope would result in, forexample, a cone-shaped or inverted cone-shaped nanowell.

Using the foregoing systems, simultaneous targeted illumination ofthousands, tens of thousands, hundreds of thousands, millions, or eventens of millions of nanowells in an array is possible. As the desire formultiplex increases, and as the density of nanowells on an arrayaccordingly increases, the ability to provide targeted illumination ofsuch arrays also increases in difficulty, as issues of nanowellcross-talk (signals from neighboring nanowells contaminating each otheras they exit the array), decreased signal: noise ratios and increasedrequirements for dissipation of thermal energy at higher levels ofdenser illumination, and the like, increase. The target waveguidedevices and optical analytical systems of the instant specificationaddress some of these issues by providing improved illumination of thewaveguides optically coupled to the arrayed nanowells.

Accordingly, the instant disclosure provides optical analytical systemscomprising an optical source, such as a laser or another suitableoptical source, and an integrated target waveguide device, such as amultiplexed integrated DNA sequencing chip, where the optical source andthe target device are optically coupled to one another.

In some system embodiments, particularly where, as described below, thetarget waveguide device comprises a heat spreading layer, the instantoptical analytical systems further comprise a heat sink, wherein theheat sink is in thermal contact with the heat spreading layer of thetarget device. The heat sink thus receives thermal energy from the heatspreading layer and thereby prevents the optical couplers on the targetdevice from overheating. Such a heat sink may optionally contain fins orthe like, in order to maximize surface area and thus heat exchange withthe surrounding environment. The heat sink may alternatively, or inaddition, contain a refrigerant, or other appropriate liquid, to furtherimprove the efficiency and heat capacity of the device. The heat sinkmay optionally still further include a fan or other such circulatingdevice for still further improvement of thermal transfer.

Target Waveguide Devices

As mentioned above, the optical analytical systems of the instantspecification comprise a target device that, in some embodiments,comprises a plurality of integrated optical waveguides to deliverexcitation energy to an array of samples within the device. The use ofintegrated optical waveguides to deliver excitation illumination isadvantageous for numerous reasons. For example, because the illuminationlight is applied in a spatially focused manner, e.g., confined in atleast one lateral and one orthogonal dimension, using efficient opticalsystems, e.g., fiber optics, waveguides, multilayer dielectric stacks(e.g., dielectric reflectors), etc., the approach provides an efficientuse of illumination (e.g., laser) power. For example, illumination of adevice comprising an array of nanowells using waveguide arrays asdescribed herein can reduce the illumination power ˜10- to 1000-fold ascompared to illumination of the same substrate using a free spaceillumination scheme comprising, for example, separate illumination(e.g., via laser beams) of each reaction site. In general, the higherthe multiplex (i.e., the more surface regions to be illuminated on thesubstrate), the greater the potential energy savings offered bywaveguide illumination. In addition, if the optical energy, for examplefrom a laser source, is efficiently coupled into the optical analyticalsystem, waveguide illumination need not pass through a free spaceoptical train prior to reaching the surface region to be illuminated,and the illumination power can be further reduced.

In addition, because illumination of samples is provided from within theconfined regions of the target device itself (e.g., optical waveguides),issues of illumination of background or non-relevant regions, e.g.,illumination of non-relevant materials in solutions, autofluorescence ofsubstrates, and/or other materials, reflection of illuminationradiation, etc., are substantially reduced.

In addition to mitigating autofluorescence of substrate materials withina target device, the coupling of excitation illumination to integratedwaveguides can substantially mitigate autofluorescence associated withan optical train. In particular, in typical fluorescence spectroscopy,excitation light is directed at a reaction of interest through at leasta portion of the same optical train used to collect signal fluorescence,e.g., the objective and other optical train components. As such,autofluorescence of such components will contribute to the detectedfluorescence level and can provide signal noise in the overalldetection. Because the systems provided herein typically directexcitation light into the device through a different path, e.g., througha grating coupler, or the like, optically connected to the waveguide inthe target device, this source of autofluorescence is eliminated.

Waveguide-mediated illumination is also advantageous with respect toalignment of illumination light with surface regions to be illuminated.In particular, substrate-based analytical systems, and particularlythose that rely upon fluorescent or fluorogenic signals for themonitoring of reactions, typically employ illumination schemes wherebyeach analyte region must be illuminated by optical energy of anappropriate wavelength, e.g., excitation illumination. While bathing orflooding the substrate with excitation illumination serves to illuminatelarge numbers of discrete regions, such illumination may suffer from themyriad complications described above. To address those issues, targetedexcitation illumination can serve to selectively direct separate beamsof excitation illumination to individual reaction regions or groups ofreaction regions, e.g. using waveguide arrays. When a plurality, e.g.,hundreds, thousands, millions or tens of millions, of analyte regionsare disposed upon a substrate, alignment of a separate illumination beamwith each analyte region becomes technically more challenging and therisk of misalignment of the beams and analyte regions increases.Alignment of the illumination sources and analyte regions can be builtinto the system, however, by integration of the illumination pattern andreaction regions into the same component of the system, e.g., a targetwaveguide device. In some cases, optical waveguides are fabricated intoa substrate at defined regions of the substrate, and analyte regions aredisposed upon the area(s) of the device occupied by the waveguides.

Finally, in some aspects, substrates used in the target waveguidedevices are provided from rugged materials, e.g., silicon, glass, quartzor polymeric or inorganic materials that have demonstrated longevity inharsh environments, e.g., extremes of cold, heat, chemical compositions,e.g., high salt, acidic or basic environments, vacuum, and zero gravity.As such, they provide rugged capabilities for a wide range ofapplications.

Waveguide devices used in the analytical systems of the presentspecification generally include a matrix, e.g., a silica-based matrix,such as silicon, glass, quartz or the like, polymeric matrix, ceramicmatrix, or other solid organic or inorganic material conventionallyemployed in the fabrication of waveguide substrates, and one or morewaveguides disposed upon or within the matrix, where the waveguides areconfigured to be optically coupled through free space to an opticalenergy source, e.g., a laser, optionally through an intervening opticalfiber, a PLC, one or more lenses, prisms, mirrors, or the like.Waveguides of the instant integrated devices can be in variousconformations, including but not limited to planar waveguides andchannel waveguides. Some preferred embodiments of the waveguidescomprise an array of two or more waveguides, e.g., discrete channelwaveguides, and such waveguides are also referred to herein as waveguidearrays. Further, channel waveguides can have different cross-sectionaldimensions and shapes, e.g., rectangular, circular, oval, lobed, and thelike; and in certain embodiments, different conformations of waveguides,e.g., channel and/or planar, can be present in a single waveguidedevice.

In typical embodiments, a waveguide in a target waveguide devicecomprises an optical core and a waveguide cladding adjacent to theoptical core, where the optical core has a refractive index sufficientlyhigher than the refractive index of the waveguide cladding to promotecontainment and propagation of optical energy through the core. Ingeneral, the waveguide cladding refers to a portion of the substratethat is adjacent to and partially, substantially, or completelysurrounds the optical core. The waveguide cladding layer can extendthroughout the matrix, or the matrix can comprise further “non-cladding”layers. A “substrate-enclosed” waveguide or region thereof is entirelysurrounded by a non-cladding layer of matrix; a “surface-exposed”waveguide or region thereof has at least a portion of the waveguidecladding exposed on a surface of the substrate; and a “core-exposed”waveguide or region thereof has at least a portion of the core exposedon a surface of the substrate. Further, a waveguide array can comprisediscrete waveguides in various conformations, including but not limitedto, parallel, perpendicular, convergent, divergent, entirely separate,branched, end-joined, serpentine, and combinations thereof. In general,a waveguide that is “disposed on” a substrate in one of the instantdevices, for example, a target waveguide device, can include any of theabove configurations or combinations thereof.

A surface or surface region of a waveguide device is generally a portionof the device in contact with the space surrounding the device, and suchspace can be fluid-filled, e.g., an analytical reaction mixturecontaining various reaction components. In certain preferredembodiments, substrate surfaces are provided in apertures that descendinto the substrate, and optionally into the waveguide cladding and/orthe optical core. As discussed above, in certain specific embodiments,such apertures are very small, e.g., having dimensions on the micrometeror nanometer scale.

The waveguides of the subject target devices provide illumination via anevanescent field produced by the escape of optical energy from theoptical core. The evanescent field is the optical energy field thatdecays exponentially as a function of distance from the waveguidesurface when optical energy passes through the waveguide. As such, inorder for an analyte of interest to be illuminated by the waveguide, itmust be disposed near enough to the optical core to be exposed to theevanescent field. In preferred embodiments, such analytes areimmobilized, directly or indirectly, on a surface of the targetwaveguide device. For example, immobilization can take place on asurface-exposed waveguide, or within a nanowell etched in the surface ofthe device. In some preferred aspects, the nanowells extend through thedevice to bring the analyte regions closer to the optical core. Suchnanowells can extend through a waveguide cladding surrounding theoptical core, or can extend into the core of the waveguide itself.Examples of using optical waveguides to illuminate analytical samples innanoscale reaction volumes are provided in in U.S. Pat. No. 7,820,983and U.S. Patent Application Publication No. 2012/0085894, which areincorporated by reference herein in their entirety.

Target Waveguide Devices with Low Numerical Aperture

Because the target waveguide devices of the instant disclosure aredesigned to be removable from an optical analytical system, and becausethe tolerances between an optical source and its associated targetwaveguide device must therefore be relatively relaxed, the opticalinput, or inputs, of the instant integrated target waveguide devices areconfigured to receive an optical signal, or signals, through free spacefrom an optical source. In particular, the optical couplers of theinstant target devices are configured for coupling from the opticalsource through free space at a distance of at least 1 mm, at least 2 mm,at least 3 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least30 mm, at least 50 mm, at least 100 mm, or even longer distances. Insome embodiments, the devices are configured for optical coupling fromthe optical source through free space at a distance of at least 5 mmMore specifically, the coupling can be at a distance of at least 10 mmEven more specifically, the coupling can be at a distance of at least 20mm.

The instant devices can be configured to receive optical energy throughfree space in a variety of ways. In particular, the dimensions, shape,orientation, composition, and other properties of the optical componentsof the devices are chosen to provide such optical coupling through freespace, as described in more detail below and in the Examples section. Insome embodiments, the optical couplers of the target device arediffractive grating couplers, although other optical couplers, such asendfire couplings, prism couplings, or any other suitable optical input,can be usefully coupled to the integrated waveguides in the instantdevices. Furthermore, the instant target waveguide devices preferablyhave multiple optical inputs, so that the optical energy is coupled intomultiple independent waveguide pathways arrayed within the device.

These and other features distinguish the instant devices and systemsfrom those typically used for optical transmission and coupling intelecommunications and other related applications, where optical sourcesare typically coupled to their targets through extremely shortdistances. Indeed, the distances typically coupled in an integratedtelecommunications optical device are on the order of 10 μm or evenless. For example, U.S. Patent Application Publication No. 2014/0177995discloses devices for optical coupling from an integrated device to anexternal optical fiber, where the outputs include couplers that comprisean integrated waveguide structure, a mirror structure, and a taperedvertical waveguide, where the vertical waveguide has apertures in therange of 0.1 to 10 μm and typical heights of 5-30 μm. These couplers,also known as vertical spot size converters, are designed for direct ornearly direct connection between the integrated waveguide structure andan associated output fiber. The devices optionally include a microlensof diameter less than 1 mm fabricated within the vertical waveguide.Another example of the direct, or nearly direct, coupling between anintegrated waveguide device and an associated target optical fiber isprovided in U.S. Patent Application Publication No. 2015/0001175, whichdiscloses the use of cylinder-shaped or sphere-shaped microlenses tofacilitate optical coupling. The lenses are fabricated with radiiroughly the same as the ˜10 μm mode size of a typical telecommunicationsoptical fiber, where the fiber is directly abutted with the microlens.These couplers are thus also designed for direct or nearly directconnection between the integrated waveguide structure and the targetfiber at the time of device manufacture.

The target devices of the instant disclosure thus comprise an opticalcoupler and an integrated waveguide that is optically coupled to theoptical coupler. In some embodiments, the optical coupler of the instantdevices is a low numerical aperture coupler, and in some embodiments,the optical coupler is a diffraction grating coupler.

Grating couplers and their use in coupling light, typically light fromoptical fibers, to waveguide devices are known in the art. For example,U.S. Pat. No. 3,674,335 discloses reflection and transmission gratingcouplers suitable for routing light into a thin film waveguide. Inaddition, U.S. Pat. No. 7,245,803 discloses improved grating couplerscomprising a plurality of elongate scattering elements. The couplerspreferably have a flared structure with a narrow end and a wide end. Thestructures are said to provide enhanced efficiency in coupling opticalsignals in and out of planar waveguide structures. U.S. Pat. No.7,194,166 discloses waveguide grating couplers suitable for couplingwavelength division multiplexed light to and from single mode andmultimode optical fibers. The disclosed devices include a group ofwaveguide grating couplers disposed on a surface that are allilluminated by a spot of light from the fiber. At least one gratingcoupler within the group of couplers is tuned to each channel in thelight beam, and the group of couplers thus demultiplexes the channelspropagating in the fiber. Additional examples of grating couplers aredisclosed in U.S. Pat. No. 7,792,402 and PCT International PublicationNos. WO 2011/126718 and WO 2013/037900. A combination of prism couplingand grating coupling of a multi-wavelength optical source into anintegrated waveguide device is disclosed in U.S. Pat. No. 7,058,261.

FIGS. 1A-1C provide a general comparison between target waveguidedevices that are coupled directly, or nearly directly, to an opticalsource with a high numerical aperture, and those, as disclosed herein,where coupling is through free space to an optical source with a lownumerical aperture. As shown in FIG. 1A, where light is coupled from anoptical fiber (100) or other optical source with high numerical apertureto a target waveguide device (110), the optical beam (102) travels arelatively short distance and thus displays a relatively small beamradius. As shown, the optical beam illuminates a grating coupler (106)that is optically connected to an integrated waveguide (108) within thetarget device. For comparison, as shown in FIG. 1B, the target waveguidedevices of the instant disclosure (e.g., 160) are illuminated by anoptical beam (152) that travels a longer distance from the opticalsource (150) and displays a larger beam radius than the system shown inFIG. 1A. The larger beam, after optionally passing through a lenselement (154) or the like, illuminates a relatively larger gratingcoupler (156) and is then launched into the optically coupled integratedwaveguide (158) associated with the coupler.

FIG. 1C illustrates an alternative embodiment of this type of opticalsystem. Specifically, in this system, one or more optical elements(e.g., 184) are positioned between an optical source (e.g., 180) and atarget waveguide device (e.g., 190). Such optical elements can serve tofocus, collimate, or otherwise modify an optical beam (e.g., 182) beforeit illuminates the target waveguide device. The optical element can, forexample, modulate the focus of the beam to more closely match thenumerical aperture (NA) of the grating coupler (e.g., 186) on the targetdevice, as would be understood by those of ordinary skill in the art.The optical element can likewise, for example, modulate the size of thefootprint of the beam on the grating coupler, as desired. As should beunderstood from this example, the NA of the optical output of theoptical source need not exactly match the NA of the input coupler on thetarget device, since an intervening lens or other optical element can beused to modulate the optical properties of the beam between the opticalsource and the target waveguide device.

In one aspect, the instant disclosure therefore provides targetwaveguide devices with one or more optical inputs that are configured tocouple light through free space from an optical source or sources. Theoptical source can be delivered to the target device through anintermediate optical component, for example through a PLC or the like,such as the PLCs disclosed in co-owned U.S. Patent Application Nos.62/133,965 and 15/072,146. According to some embodiments, the numericalaperture (NA) of the optical inputs in the target waveguide devices ismodulated in order to facilitate and optimize coupling into the targetdevice in various ways. As is understood by those of ordinary skill inthe optical arts, NA is related to the range of angles within whichlight, in particular a light source approximating a Gaussian light beam,can be accepted or emitted from a lens, a fiber, a waveguide, a gratingcoupler, or the like. It is a dimensionless value that, in the case of aGaussian beam impinging on an objective lens, can be calculated usingthe following equation:

NA=n sin θ_(max)

where n is the index of refraction of the medium through which the beamis propagated and θ_(max) is the maximum acceptance angle of the lens.This angle corresponds to the half-angle of the lens's acceptance cone,i.e., the cone of light capable of entering or exiting the lens.

In the case of a multi-mode optical fiber, the numerical aperturedepends on n_(core), the refractive index of the core, and n_(clad), therefractive index of the cladding, according to the following equation:

NA=√{square root over (n _(core) ² −n _(clad) ²)}

The NA of an optical device, such as a fiber or an integrated waveguide,thus can depend on the optical properties of the materials used tofabricate the device (e.g., the core and the cladding of a fiber orwaveguide) and the size and geometry of the device. The NA also dependson the wavelength of light being propagated through the device. Itshould thus be understood that the NA of a particular optical device canbe usefully modulated to obtain suitable behavior of the device for aparticular application and purpose.

From a practical standpoint, the NA of a given optical device can alsobe determined empirically, for example by measuring the characteristicsof propagated light emitted by the device at a certain distance from theend of the device, for example using a direct far field scanneraccording to specification EIA/TIA-455-47. Such measurements provideempirical values of the mode field diameter (MFD), effective area, andnumerical aperture of the optical device. In the case of a single-modefiber, the MFD is related to the spot size of the fundamental mode andrepresents a far-field power distribution of the optical output of thefiber. The relationship between NA and MFD for a Gaussian beam isprovided by the following equation, where λ is the wavelength ofpropagated light:

${MFD} = {\frac{2}{\pi} \times \frac{\lambda}{NA}}$

Table 1 shows the relationship between NA and beam diameter for light of532 nm, where the Gaussian beam profile is truncated at three differentpower levels: 1/e², 1/e³, and 1/e⁴. The listed beam diameters at a powertruncation of 1/e² correspond to the MFD of the beam for each value ofNA. The listed beam diameters at a power truncation of 1/e³ provide auseful estimation in designing the size of an optical coupler on atarget device. More specifically, a coupler of the cross-sectional sizeshown in this column will capture most of the energy from thetransmitted beam.

As is known in the art, single mode fiber devices are commonly used in avariety of optical devices for the transmission and coupling of opticalsignals, particularly in the telecommunications industry. Such devicestypically display NA values of 0.12 or greater. As shown in Table 1,such NAs, for example NAs of 0.12 and 0.13, result in relatively narrowbeam sizes at this wavelength of light: 2.82 μm and 2.61 μm,respectively. By comparison, a Gaussian beam of 532 nm light with an NAof 0.01 displays a beam size of approximately 34 μm—10 times larger.FIG. 2A shows the 2-dimensional profile of such a Gaussian beam (NAequal to 0.01). As just noted, the beam size is determined by thetruncation of beam profile at the 1/e² power level.

TABLE 1 Power-truncated beam profiles for light of 532 nm as a functionof NA. NA 1/e² (μm) 1/e³ (μm) 1/e⁴ (μm) 0.13 2.61 3.91 5.21 0.12 2.824.23 5.64 0.05 6.77 10.16 13.55 0.015 22.58 33.87 45.16 0.01 33.87 50.8067.74 0.005 67.74 101.60 135.47

It should also be understood that the diameter of a Gaussian beam willvary along the beam axis due to beam divergence. More specifically, fora divergent Gaussian beam propagated in free space, the beam radius, w,varies as a function of distance, z, along the length of the beam axisaccording to the equation:

${w(z)} = {w_{0}\sqrt{1 + \left( \frac{z}{z_{R}} \right)^{2}}}$

where w₀ is the minimum beam radius, i.e., the “waist radius”, thatoccurs at a particular location along the beam axis known as the “beamwaist”, z is the distance from the beam waist along the beam axis, andz_(R) is the Rayleigh length, a constant for a given beam that dependson the waist radius and the wavelength of light, λ, according to:

$z_{R} = \frac{\pi w_{0}^{2}}{\lambda}$

Accordingly, at a distance along the beam axis of z_(R) from the beamwaist, the beam radius is equal to w₀√{square root over (2)}. In view ofthe above, it also follows that the Rayleigh length and the numericalaperture are related to one another according to the following equation:

${NA} = \frac{w_{0}}{z_{R}}$

The above parameters are illustrated graphically in FIG. 2B, whichrepresents a divergent Gaussian beam of radius w.

In accordance with the above description, lenses, fibers, and waveguideswith relatively large NA values are typically used to illuminate targetsurfaces over short distances through free space, and the spot size ofsuch illumination is typically small. These distinctions are apparent inthe exemplary systems illustrated graphically in FIGS. 1A and 1B.Specifically, the optical device (100) of the system shown in FIG. 1A(e.g., an optical fiber) has a high NA, and is best suited forilluminating a small-diameter coupler at close proximity to the targetwaveguide device (110). By comparison, the optical source (150) and lens(154) of the system shown in FIG. 1B has a low NA, and, as describedherein, is well suited for illuminating a large-diameter coupler at alarge free-space coupling distance. As mentioned above, FIG. 1C shows analternative design that permits the optical footprint of the output beamto be re-imaged with a target magnification, for example using anintervening optical element, to provide a beam waist of a preferred sizeat the surface of the target device. It should be further noted herethat the illustrations provided throughout the disclosure are notnecessarily intended to represent accurately the dimensions, angles, orother specific design features of the devices illustrated, in particularany representation of divergence angles, beam radii, layer thicknesses,waveguide bend radii, specific routing paths, and so forth.

Free-space coupling, as disclosed in the devices and systems herein,provides several advantages relative to the direct, or nearly direct,coupling typically used in telecommunications and related systems.First, coupling through free space avoids near-surface fiber tip to chipoperation and is thus much easier for installation and operation andmuch less vulnerable to chip-surface dust and contamination and tipdamage due to mis-operation of optical analytical systems with removabletarget waveguide devices. Second, as illustrated in FIGS. 1B and 1C,coupling with low NA delivery devices through free space allows largerbeam diameters on the target waveguide device, thus relieving thermalconstraints on the target chip due to the injection of high laser power.Third, larger grating coupler size also greatly alleviates opticalsource-to-chip alignment difficulties and minimizes the impact of dustand other contaminants on the coupler surface. Fourth, free-spacecoupling allows easier chip packaging solutions for the target chip,which, for example in a multiplexed DNA sequencing chip, needs toaccommodate all the packaging interface requirements such as electrical,thermal, mechanical, and fluidics components. Use of larger couplers isparticularly advantageous in applications where surface-area constraintsare not of overriding importance, for example in some applications usingcommercial CMOS chips. In view of the above, it should be apparent thatthe input NA of the instant target waveguide devices can thus bemodulated in order to improve and optimize optical coupling from anassociated optical source.

Accordingly, the instant disclosure provides target waveguide deviceswith one or more optical inputs that are configured to couple lightthrough free space from an optical source or sources through ahigh-efficiency input coupler. Such devices can optionally compriseadditional features, for example further integrated waveguides,preferably in an array, and a plurality of nanowells optically coupledto the waveguide or array of waveguides. As described above, an array ofnanowells in optical connection with an excitation source can beusefully employed, for example, in the performance of highly-multiplexedDNA sequencing reactions using fluorescently-labeled nucleotidereagents.

The free-space coupling of optical energy into the instant targetdevices is preferably achieved through the use of a high-efficiency,low-NA grating coupler. An exemplary grating coupler is illustrated inFIG. 3A. Such couplers are conveniently prepared using standardsemiconductor processing techniques on, for example, a silicon chip orother suitable substrate (320). The grating typically includes a bottomcladding layer (324), a waveguide core layer (308), and a top claddinglayer (322), where the core layer has a higher refractive index than thecladding layers, so that light injected into the core is propagated bytotal internal reflection at the core/cladding boundaries. A gratingstructure (306) is created in the waveguide core, typically during thefabrication process, with a desired duty cycle (312), etch depth (314),and grating period (316), such that optical energy (302) incident on thesurface of the grating can enter the grating and be efficientlypropagated down the waveguide core. FIG. 3B shows a variant of thegrating coupler of FIG. 3A, where the waveguide core is etched as shownto provide a “blazed” coupler region (326).

The detailed grating coupler structures and shapes can be varied in anumber of ways to improve the coupling efficiency. For a simple binarygrating coupler, the structure can be etched from the top only, asillustrated in FIGS. 3A, 3B, and 3E, or from the bottom only, asillustrated in FIG. 3C. Alternatively, the structure can be double-sidedetched from both the top and the bottom, as illustrated in FIGS. 3D and3F. Moreover, an overlay layer can be added to the structure to theincrease the teeth height, as illustrated in the grating couplerstructures of FIGS. 3E and 3F, thus further improving the couplingefficiencies of the gratings. The period of the grating coupler can befixed as a uniform grating, or it can be “chirped” with a certainfunction, by fabricating the teeth with a non-uniform period, to bettermatch the Gaussian beam profile, as illustrated in the grating coupler(346) illustrated in FIG. 3G, thus improving the coupling efficiency.Alternatively, or in addition, a bottom reflective layer (370) can beadded to the structure, as illustrated in FIG. 3H, to reflect thedown-coupling light and thus to improve coupling efficiency.

In some embodiments, the grating period of the instant grating couplersis in the range from 300 nm to 1000 nm. In more specific embodiments,the grating period is in the range from 300 nm to 500 nm and from 300 nmto 400 nm. In even more specific embodiments, the grating period is from340 nm to 380 nm and can in some embodiments be approximately 355 nm. Inother even more specific embodiments, the grating period is from 300 nmto 340 nm and can in some embodiments be approximately 315 nm.

In some embodiments, the etch width of the instant grating couplers isin the range from 150 nm to 500 nm. In more specific embodiments, theetch width is in the range from 150 nm to 400 nm. In even more specificembodiments, the etch width is in the range from 150 to 300 nm and canin some embodiments be approximately 185 nm.

In embodiments, the etch depth of the instant grating couplers is in therange from 30 nm to 200 nm, is in the range from 50 nm to 150 nm, or isin the range from 50 nm to 100 nm. Specifically, the etch depth can beapproximately 68 nm. In some embodiments, the etch depth is in the rangefrom 30 nm to 80 nm. Specifically, the etch depth can be approximately55 nm.

The thickness of the waveguide core of the instant grating couplers ispreferably optimized for single-mode operation using light of a desiredwavelength. The optimal core thickness (“d”) can accordingly beestimated using the following relationship:

$V = \left. {{\frac{\pi \; d}{\lambda}\sqrt{n_{core}^{2} - n_{clad}^{2}}} < \frac{\pi}{2}}\Rightarrow{d < {\frac{\lambda}{2} \cdot \frac{1}{\sqrt{n_{core}^{2} - n_{clad}^{2}}}}} \right.$

For a typical waveguide construction, with a silicon nitride core (e.g.,n_(core)≈1.9085) and a silicon dioxide cladding (e.g., n_(clad)≈1.46), acore thickness of about 217 nm is optimal for light with wavelength of532 nm, and a core thickness of about 225 nm is optimal for light withwavelength of 552 nm. Where the refractive index of the waveguide coreis increased, for example by using a titanium oxide core, or the like,optimal core thicknesses can be significantly smaller For example, wheren_(core)=2.55 and n_(clad)=1.46, optimal core thicknesses of 127 nm(@532 nm) and 132 nm (@552 nm) can be estimated. In view of the above,the waveguide core thickness of the instant grating couplers can rangefrom about 100 nm to about 300 nm. More specifically, the waveguide corethickness can range from about 100 nm to about 150 nm, and even morespecifically from about 125 nm to about 135 nm. In some embodiments, thewaveguide core thickness can range from about 150 nm to about 250 nm,more specifically from about 200 nm to about 240 nm, and even morespecifically from about 215 nm to about 230 nm. In some embodiments, thewaveguide core thickness can be approximately 180 nm.

In embodiments, the waveguide core refractive index of the instantgrating couplers is in the range from 1.9 to 3.5 and more specificallyis approximately 1.9. In some embodiments, the waveguide core refractiveindex is in the range from about 2.4 to about 2.7, more specificallyfrom about 2.5 to about 2.6. In embodiments, the top cladding thicknessof the instant grating couplers is in the range from 250 nm to 1000 nm,more specifically is approximately 280 nm. In embodiments, the bottomcladding thickness of the instant grating couplers is in the range from2 μm to 10 μm and more specifically is approximately 2.1 μm. Inembodiments, the cladding refractive index of the instant gratingcouplers is in the range from 1 to 2 and is more specificallyapproximately 1.47. It should be understood that refractive indices arepreferably specified for a given material at the wavelength of lightbeing transmitted through the material, as would be understood by thoseof ordinary skill in the art.

In device embodiments comprising a reflective layer, the reflectordistance (from coupler bottom to the reflector) of the devices can be inthe range from 250 nm to 500 nm and can be more specificallyapproximately 260 nm.

As mentioned above, the NA of the instant target waveguide devices canbe modulated in order to improve coupling from the optical sourcethrough free space. In embodiments, the NA of the target waveguidedevice is modulated to match the NA of the optical source. According tosome embodiments, the optical input of the instant devices has anumerical aperture of no more than 0.1, no more than 0.08, no more than0.05, no more than 0.03, no more than 0.02, no more than 0.01, no morethan 0.005, or even lower. In some embodiments, the numerical apertureis no more than 0.05. In specific embodiments, the numerical aperture isno more than 0.015.

As should be apparent from the comparison shown in FIGS. 1A and 1B,although the NA of traditional optical sources and targets (e.g., 100and 110) is significantly higher than that of the optical sources andtargets used in the instant systems (e.g., 150 and 160), the surfacearea or “footprint” illuminated on the instant target devices is larger.(For example, compare the size of grating couplers 106 and 156.) Asnoted above, larger optical footprints can be advantageous inter alia inminimizing heating of the target device and/or in simplifying alignmentof the optical source and the target device. In particular, the powerintensity of the transmitted light is much lower than it would be if thelight were transmitted in a more focused beam.

The exact spot size of light delivered to a target waveguide devicewill, of course, depend both on the NA of the optical outputs of theoptical source and the free space distance between the optical sourceand the target device. In embodiments, the target waveguide device isdesigned with a coupler size that matches the spot size of illuminationfrom the optical source. In embodiments, the coupler size of the targetdevice is at least 100 μm², at least 144 μm², at least 225 μm², at least400 μm², at least 625 μm², at least 900 μm², at least 1600 μm², at least2500 μm², at least 4900 μm², at least 10,000 μm², or even larger.

In other embodiments, the coupler size of the target device is at most250,000 μm², at most 62,500 μm², at most 22,500 μm², at most 10,000 μm²,at most 6400 μm², at most 3600 μm², or at most 2500 μm².

In specific embodiments, the coupler size of the target device is from100 μm² to 250,000 μm², from 225 μm² to 62,500 μm², from 400 μm² to22,500 μm², from 625 μm² to 10,000 μm², from 900 μm² to 6400 μm², orfrom 1600 μm² to 3600 μm².

In embodiments, the above-described illuminations are achieved at afree-space distance between the optical source and the target device offrom 1 mm to 100 mm More specifically, the free-space distance can befrom 2 mm to 90 mm, from 5 mm to 80 mm, from 10 mm to 60 mm, or evenfrom 20 mm to 50 mm.

It also follows from the above description that the instant targetwaveguide devices are capable of receiving relatively high levels ofoptical energy from an optical source due to the relatively large spotsizes illuminated on the target device. Accordingly, in embodiments, thetarget device is configured to receive optical energy with power percoupler of at least 1 mW, at least 2 mW, at least 3 mW, at least 5 mW,at least 10 mW, at least 20 mW, at least 30 mW, at least 50 mW, at least100 mW, or even higher per coupler. In specific embodiments, these powerlevels are achieved at a free-space distance of at least 10 mm.

According to another aspect of the disclosure, it can be desirable tomodulate the design of the integrated waveguides in the target waveguidedevice in order to improve the coupling between the optical source andthe target waveguide device. In particular, it can be desirable tomodulate the composition and shape of the integrated waveguides toachieve these effects. For example, it is known in the field of opticsthat mismatches between the mode sizes and effective indices between thehighly confined mode of an integrated optical waveguide and the largediameter mode of an optical fiber input can result in coupling losses ifnot addressed. It can therefore be advantageous to taper the waveguidegeometry or otherwise vary the waveguide structure and/or composition inorder to improve the behavior and efficiency of the device, particularlyin transitions between confined and unconfined optical modes. Suchvariation in structure and composition can include, for example,modulation of cladding composition and geometry or modulation of corecomposition and geometry. In particular, core cross-sectional geometrycan be modulated to improve coupling efficiencies. These and otherfeatures can be modeled and tested using widely available commercialsoftware to predict and optimize the photonic properties of the devicesprior to their fabrication.

In some applications, it can be advantageous to vary the optical poweremitted from each optical output of an optical source according to thespecific requirements of the target device, for example to compensatefor propagation losses as the light passes through the targetwaveguides. Such approaches are described in co-owned U.S. PatentApplication Nos. 62/133,965 and 15/072,146. Other advantageous featuresand designs that can optionally be included in the instant targetwaveguide devices are disclosed in U.S. Patent Application PublicationNos. 2014/0199016 and 2014/0287964, which are incorporated by referenceherein in their entireties.

The waveguide devices and systems of the instant disclosure can befurther distinguished from those typically used in transmitting opticalsignals in telecommunications applications. In particular, the instanttarget waveguide devices are designed for use with higher intensityoptical energy, and they are designed to transmit that energy for muchshorter distances. In addition, the wavelengths of light transmitted bythese devices are suitable for use with the optically active reagentscommonly used in biological assays. These wavelengths are generallysignificantly shorter than those used for telecommunications purposes.In particular, the optical illumination used in DNA sequencing reactionswith fluorescently-labeled DNA reagents, is typically in the visiblerange, most commonly in the range from 450 nm to 650 nm. The waveguidesand other components of the target devices and systems disclosed hereinare therefore preferably designed and scaled to transmit optical energyefficiently in the visible range. In some embodiments, the wavelengthsrange from about 400 nm to about 700 nm. In more specific embodiments,the wavelengths range from about 450 nm to 650 nm or even from about 500nm to about 600 nm. In some specific embodiments, the wavelengths arefrom about 520 nm to about 540 nm, for example, approximately 532 nm. Inother specific embodiments, the wavelengths are from about 620 nm toabout 660 nm, for example, approximately 635 nm or 650 nm. In stillother specific embodiments, the devices are designed for optimaltransmission of light having wavelengths from about 540 nm to about 560nm, for example, approximately 552 nm. In some embodiments, multiplewavelengths of visible light can be transmitted within the devicessimultaneously. A silicon nitride waveguide device, including anintegrated grating coupler, for the transmission of visible wavelengthshas recently been reported. Romero-García et al. (2013) Opt. Express 21,14036. Accordingly, in some embodiments, the waveguide core material isa silicon nitride. In other embodiments, the waveguide core material isa material having an even higher refractive index at the wavelengthsused in the instant device, for example a titanium oxide, such astitanium dioxide (TiO₂). Such higher refractive index materials alsopreferably display low autofluorescence.

The grating couplers of the instant devices may in some embodiments bebeam focusing couplers. In particular, in order to avoid the long taperassociated with the reduction of mode size from the large footprint,low-NA grating couplers (where mode size can be, for example, 50 μm) toa mode size effective in illuminating nanoscale sample wells (forexample, 0.5 μm), the shape of the coupler can be changed fromrectangular (as viewed from the top) to tapered (as viewed from the top)to form an ultracompact focusing grating coupler. The top view of onesuch exemplary coupler design is illustrated in FIG. 3I.

Beam focusing couplers may bend the grating lines to be a series ofconfocal ellipses with the focal point located at the grating-waveguideinterface. Therefore, the optical mode can be directly focused from thegrating to the waveguide in a much smaller distance, in some cases onthe order of several hundred microns. As illustrated in FIGS. 3J and 3K,which are also top views of the couplers, the transition region betweenthe grating coupler and the integrated waveguide core can, for example,be a tapered waveguide (FIG. 3J) or a slab waveguide (FIG. 3K). In eachcase, the curved grating lines focus the light into the aperture of theintegrated waveguide. Also identified in these figures are two relevantparameters—focal length and defocus—that are of importance in the designof a beam focusing coupler. Furthermore, as shown with the slabwaveguide transition region of FIG. 3K, the aperture of the integratedwaveguide targeted by the grating coupler can be tapered to a widerwidth in order to achieve optimal coupling. FIG. 3L illustrates across-sectional profile of an exemplary focusing grating coupler,indicating preferred chemical compositions of the various layers andexemplary dimensions of the various features.

In this regard, for some target waveguide device embodiments, where thecoupler is a focusing grating coupler, the focusing coupler focal lengthcan be in the range from 150 μm to 500 μm and can be more specificallyapproximately 170 μm. In some embodiments, the focusing coupler defocusof the instant grating couplers is in the range from 0 to 10 μm and ismore specifically approximately 0 μm. In some embodiments, the focusingcoupler aperture width is in the range from 1 μm to 5 μm and is morespecifically approximately 3 μm. In some embodiments, the waveguidetaper length of the focusing grating couplers is in the range from 50 μmto 200 μm and is more specifically approximately 75 μm. In someembodiments, the coupling angle of the instant grating couplers is inthe range of 10 degrees+/−2 degrees. In a specific design, the coupleris a slab coupler with focal length=150 μm, defocus=0, and aperturewidth=3 μm.

Furthermore, the above design features and parameters of a targetwaveguide device can be combined, in any suitable way, to maximize thecoupling efficiency. The design and fabrication of the above-describedstructures is within the skill of those of ordinary skill in the art.Exemplary grating couplers are described in the references providedabove. Other exemplary waveguide devices with grating couplers,including focusing couplers and couplers with reflective metalliclayers, have also been reported. See, e.g., Waldhäusl et al. (1997)Applied Optics 36, 9383; van Laere et al. (2007) J. Lightwave Technol.25, 151; van Laere et al. (2007) DOI: 10.1109/OFC.2007.4348869 (OpticalFiber Communication and the National Fiber Optic Engineers Conference);U.S. Pat. No. 7,283,705. It should be understood, however, that thecouplers disclosed in these references are typically designed foroptimal coupling from high-NA optical sources, not from low-NA opticalsources.

Target Waveguide Devices with Alignment Features

In some embodiments, the target devices and systems of the instantdisclosure include features that provide free-space coupling between anoptical source and a target device while maintaining alignment of thecomponents to sub-micron accuracy in space, including angle tolerances.Disturbances communicated to the analytical system from the mountingsuch as shocks and vibrations may cause alignment errors that aresubstantial on the submicron scale. Pneumatic isolation, which has beenused in some prior art analytical systems, is physically large, andexpensive, in order to reject these perturbations passively. Analternative to such passive approaches is the use of an active rejectionby estimation of an alignment error, and commanding a correction, andpossibly iterating depending on the particular response of the physicalservo system. This active rejection of vibration can be small,inexpensive, and highly effective: however, this active rejectionrequires an error signal. On the time scales of interest, the imagecorrelation approaches used in some prior art instruments to estimate anerror are insufficiently fast. Hill climbing based on a dither (orperturb and observe) require higher bandwidth, more expensive actuators,or are insufficiently fast.

The dynamic alignment approach disclosed herein involves one or morealignment features that can be inexpensively incorporated into a targetwaveguide device within an optical analytical system. Such alignmentfeatures are used in combination with an alignment detector, such as analignment camera, within the analytical system to provide a continuousestimate of alignment error, thus enabling an inexpensive actuation anddetection system.

In some embodiments, the alignment features take the form of additionalgrating couplers, which may or may not be the same design as the gratingcouplers used to couple the main pump power into the device. The gratingcouplers couple input optical signals into associated alignmentwaveguides. They can be arranged in at least one, often 2, and sometimesmore locations to better estimate magnification, roll, and other errors.The alignment structures detect the light from one or more alignment or“outrigger” light beams that are directed toward the target waveguide.The alignment light beams typically emanate from the same optical sourceas the one or more sample excitation light beams (i.e., the light beamstargeting the analytical samples), so that the position of the alignmentbeams can be used as a proxy for the position of the one or more sampleexcitation light beams.

The input couplers of the alignment waveguides direct coupled light fromdesignated beams to designated output couplers, which may or may not begrating couplers. These output couplers should be readilydistinguishable from one another, so that the output power can beuniquely estimated for each. For example, where a low NA external camerais used as a detector, the spacing can be ˜150 μm.

The output estimated for each output device can then be combined,typically with a simple formula, to form what is designated a trackingerror signal (TES). This TES, for each dimension of interest, can thenbe converted into a command to counter the present state ofmisalignment. An exemplary arrangement of alignment couplers, togetherwith their associated alignment waveguides and output couplers, andsample excitation couplers, together with their associated sampleexcitation or “sequencing” waveguides, is shown in FIG. 4A. As shown,this exemplary target waveguide device includes two triads of alignmentwaveguides, shown as the top three coupler/waveguide combinations andthe bottom three coupler/waveguide combinations in the drawing. Eachtriad of alignment couplers is illuminated by a single optical input,illustrated as a circular shaded region in the drawing, so that theportion of light passing through each of the waveguides depends on thealignment of the optical input with each alignment coupler. The outputfrom the alignment waveguides, designated A1, B1, and C1 for the toptriad of alignment waveguides and A2, B2, and C2 for the bottom triad ofalignment waveguides, is monitored by a camera or other suitablealignment detector device to generate a TES. If the optical source andtarget device move relative to one another during a measurement, it isapparent that the TES generated by each trio of waveguides will change.Alignment can be maintained, and misalignment can be reversed, bymonitoring the TES values. Each triad of alignment input and outputcouplers and their associated alignment waveguides should be considereda single alignment feature for purposes of this disclosure.

Also shown in the device of FIG. 4A are sample excitation couplers, inthis case fabricated between the two triads of alignment couplers. Thesample excitation couplers are used to deliver optical energy from theinput beams, which are identified in FIG. 4A as circular shaded regionswithin each coupler, to the analytical nanoscale samples within thedevice, typically through a fanout region of the device. The fanoutregion splits the incoming excitation signal into a larger number ofsplit waveguides for delivery to the arrays of nanoscale sample wells inthe device. One or more of the sample excitation waveguides associatedwith each input coupler can additionally be used to monitor power levelsof optical energy input into the sample excitation input coupler. Thesepower monitoring waveguides can deliver their optical signals to anoutput coupler for monitoring by a power output detector. In someembodiments, for example as shown in the device of FIG. 4A, the poweroutput monitoring couplers, identified as circular shaded regions at theend of the “sequencing WGs” in FIG. 4A, are located near the alignmentwaveguide output couplers. In these embodiments, a single detector, forexample a single camera, can be used to monitor both the alignmentwaveguide signals and the sample excitation waveguide power outputmonitoring signals simultaneously.

The input couplers of the alignment waveguides and the input coupler ofa sample excitation waveguide (labeled as a “low NA input coupler”) areshown in closer detail in FIG. 4B. The optical input for the alignmentfeature in this exemplary device is a 1% beam, that is, the alignmentbeam carries about 1% of the power of all of the combined beams reachingthe device. The optical input for the sample excitation coupler is afull-power beam, also known as a “sequencing beam” or a “pump-powerbeam”. The footprints illuminated by these beams are illustrated asshaded circles in FIG. 4A and as open circles in FIG. 4B. Approximatedimensions of the exemplary input couplers are also shown in FIG. 4B.

Another exemplary arrangement of alignment features in a targetwaveguide device is illustrated schematically in FIG. 4C. This deviceincludes two “patterned”, or “stipled”, regions (460) that can serve asalignment features. These features can work independently of, or inaddition to, the alignment features described above in FIGS. 4A and 4Band as also shown in the device of FIG. 4C. The patterned regions on thedevice of FIG. 4C can be illuminated by alignment beams, which areidentified in the drawing as shaded circles (470). As previouslymentioned, the alignment beams preferably carry approximately 1% of thepower of the other beams. The illuminated patterned regions can thus beobserved and monitored by a camera or other detector device within theanalytical device in order to establish and/or maintain alignment of theoptical source and the target waveguide. As just mentioned, the targetdevice of FIG. 4C also includes two of the above-described alignmentfeatures, which comprise triads of alignment input couplers (462), theirassociated alignment waveguides (464), and their associated alignmentoutput couplers (466). The alignment output couplers are typically highnumerical aperture output couplers, which may be monitored from above byan alignment detector, such as an alignment camera, to facilitatealignment of the optical source and target waveguide device.

FIG. 4C also shows four shaded circles (472) representing the spotsilluminated by sample excitation beams from an optical source. Thesefull-power beams are coupled into the device through free space,preferably using low numerical aperture couplers, as described in detailelsewhere in the disclosure. As shown in the drawing, in this embodimentof the target device, the couplers direct the input optical energy froman optical source into tapered integrated waveguides which are directedthrough “fanout” regions to split the sample excitation beams into alarger number of split sample excitation waveguides, in this case 10split waveguides for each input beam. The split waveguides ultimatelydeliver the input optical energy to nanoscale sample wells arrayed onthe device. In the device of FIG. 4C, one of the 10 split waveguidesassociated with each coupler is directed to an output coupler (474) toserve as a power monitoring coupler, as described above. This couplercan be observed by an external detector, such as a detector camera, tomonitor power levels passing through the excitation waveguides. Thepower monitoring couplers can provide immediate feedback to the systemif the power output of an optical source changes during a measurement,or if alignment is lost between the optical source and the targetwaveguide device.

FIG. 4D illustrates another exemplary target waveguide device (480).This device includes an input coupling region (481) in the lower leftcorner of the device and a large arrayed nanoscale sample well region(490) in the main central upper portion of the device. The inputcoupling region can further include alignment features, as described indetail above. FIG. 4D also illustrates two sample excitation waveguidepathways, one starting at low NA input coupler 482, and the otherstarting at low NA input coupler 484. Input sample excitation beams arecoupled into these pathways and directed to the nanoscale sample wellseither through the top fanout region (483) for input coupler 482 orthrough the bottom fanout region (485) for input coupler 484. Within thefanout regions, the excitation waveguides are split multiple times tocreate an array of split excitation waveguides to deliver optical energyto the nanoscale sample wells. As described in detail in co-owned U.S.Patent Application Nos. 62/133,965 and 15/072,146, the different pathlengths encountered by optical energy that is input into the differentcouplers, and thus the different propagation losses suffered by thedifferent excitation waveguide pathways, can be compensated by adjustingthe power levels of optical inputs from the different couplers or bymodulating the optical signals in other ways. The example of FIG. 4Dalso illustrates that the nanoscale samples can optionally be excited byoptical energy transported through the same excitation waveguides fromtwo different directions simultaneously. As shown in this drawing, lightdelivered from input coupler 482 and light delivered from input coupler484 can be directed to the same nanoscale sample wells through theirassociated arrayed waveguides from opposite directions, if desired.

FIG. 4E illustrates an optical analytical system of the instantdisclosure, including a target waveguide device with at least one of thealignment features described in this section. The system comprises anoptical source consisting of one or more lasers, a beam powercontroller, and a “light brush”, which may correspond to one of theoptical delivery devices of co-owned U.S. Patent Application Nos.62/133,965 and 15/072,146. The system also comprises an alignmentcamera, an integrated detector component comprising an array of “pixels”for detecting optical outputs from nanoscale sample wells arrayed acrossthe target device, and a “sensor readout” component that receives andanalyzes signals from the detector. An optical beam or beams emitted bythe lasers and passing through the beam power controller and light brushis represented as a thick arrow that illuminates an input coupler on thetarget device. The optical input is coupled into the device and isdirected to one or more integrated waveguides within the device, asindicated by the smaller arrow. The optical input can optionally bedirected to one or more alignment waveguides and/or one or more powermonitoring waveguides. The alignment camera in this drawing is shownreceiving optical outputs indicated in the drawing by even smallerarrows, from output couplers at the far end of the device. Thesecouplers could be used to output light from the alignment waveguidesand/or the power monitoring waveguides. It should also be understoodthat the alignment camera can, in addition or alternatively, receiveoptical signals from other alignment features such as one or morepatterned regions, fiducials, or other reference marks on the surface ofthe target device. Optical energy traveling through the sampleexcitation waveguides illuminates samples in the arrayed nanowells, andfluorescence emitted from the samples is directed to appropriatelyaligned pixels in the detector layer, where the output signal ismeasured.

FIG. 4F illustrates in graphic form how the light brush of FIG. 4E canbe aligned with the target waveguide device that is disclosed herein,using any of the alignment features described above. Specifically, thisfigure illustrates the degrees of freedom that can be monitored andadjusted during the alignment of an optical source and a target device.As shown in the drawing, the airplane symbolizes three dimensions ofrotation relative to the target device, and the “cell surface”corresponds to the surface of the target device. In addition to therotational motions indicated in the drawing as pitch, yaw, and roll (orpattern rotation), the light brush and target device can move relativeto one another in the x, y, and z coordinate space. Two of these motionsare shown in the drawing as “along track” (AT) and “cross track” (CT)motions. Not shown in the drawing is an up and down motion to vary thedistance between the light brush and target waveguide. As shown in theinset drawing, rotation on the “roll” axis causes the input beams topivot around a particular axis. In this specific example, the lightbrush provides 12 separate input “beamlets”. The two beamlets at eachend of the illumination pattern are low-power alignment beamlets. Theirtargets on the device are illustrated as smaller circles in the line ofinput couplers on the surface of the device.

Accordingly, as described above, the alignment features of the instantdisclosure can be arranged in various ways for various purposes. Forexample, as described above, they can be arranged to normalize forincident power. As also described above, the alignment features can beused as pump power grating couplers, at the expense of some efficiency.In addition, the output monitoring devices can be grating couplers, orcan be other devices that are configured to redirect light towards analignment detector, such as a camera.

Furthermore, the light coupled into the alignment grating couplers canbe of the same wavelength as the pump power, but need not be. Likewisepolarization, input (and output) angles can differ from the pump gratingcouplers, as desired. The light in the alignment grating couplers can beeither coherent or incoherent with the pump grating couplers.

Accordingly, in some specific embodiments, the alignment feature cancomprise one or more waveguides. In more specific embodiments, thealignment feature can comprise a plurality of low-power waveguide tapsor a high-power beam tap. In other embodiments, the alignment featurecan comprise a reference mark, for example a fiducial or other type ofpatterned region. The use of reference marks in the alignment ofdifferent components of an optical analytical system is well known inthe art of printed circuit board manufacture and computer vision. See,for example, U.S. Pat. Nos. 5,140,646 and 7,831,098. The positionalinformation obtained through monitoring the alignment features of theinstant devices by an alignment detector can be used by the opticalsystem to position the optical source and the target device relative toone another prior to the start of an analytical assay. The positionalinformation can further be used during the course of an assay tomaintain the position of the optical source and the target devicedynamically through a feedback loop, as would be understood by those ofskill in the art.

Target Waveguide Devices with Improved Power Handling

In some embodiments, the target waveguide devices of the instantdisclosure comprise grating couplers with improved power handlingcapacity. In particular, one key factor limiting the amount of opticalpower that can be coupled through a grating coupler is the peak localtemperature rise in the vicinity of a focused light beam of high opticalpower density. With parameters reasonable for optical couplingperformance and typical materials and designs, the local temperature ina region below the coupler can quickly reach levels that are likely toimpair performance or cause physical damage, even with moderate inputpower (e.g., potentially much less than 1 W).

Indeed, while various examples exist of grating couplers as an interfacebetween free-space or fiber optic inputs and waveguides inmicrofabricated integrated photonic circuits, issues may arise when suchcouplers are used to transmit substantial amounts of optical power.While perfect coupling efficiency is unattainable, and with the bestreported coupling efficiencies in the range of 50% (i.e., −3 dB), asubstantial fraction of incident power is not coupled into thewaveguide. Even if a substantial portion of the uncoupled power isreflected or scattered away from the vicinity of the coupler, however,some local absorption is inevitable. With increasing input power,temperature in the vicinity of local absorption for a tightly-focusedbeam may rise to levels that may impair coupler performance or causephysical damage.

As described herein, however, by reducing the local thermal resistancebetween a limited absorbing region and the bulk of the microfabricatedcomponent, higher input power can be coupled without damage orimpairment of coupler performance. For a fluorescence application, thismeans that greater pump intensity can be utilized to improvesignal-to-noise performance, and an increased area or number of samplesites can be interrogated. In addition, for the instant target devicesand systems, where a plurality of input ports can be required due tothermal limitations, allowing more power per input port allows thenumber of input ports to be reduced, thus simplifying the optical systemand the associated target device.

Accordingly, in the grating couplers of the instant target devices, alayer of material with relatively high thermal conductivity can befabricated below the grating in order to improve the lateral heattransfer within the device and thus reduce peak temperatures.

If the design of the coupler includes a reflection layer below thegrating (optionally with some bottom cladding material in between) inorder to improve coupling efficiency, then the conductive layer can belocated immediately below and in contact with the layer of material thatforms the reflection layer interface with the bottom cladding. Dependingon materials, the interface between the conductive layer and the bottomcladding below the coupler can itself form the reflection layer, e.g.,in the case of an interface between SiO₂ and Al for visible-wavelengthapplications.

In order to serve as an effective heat spreader, thethermally-conductive layer should have a thickness greater than requiredfor purely optical purposes (which can in specific embodiments be onlyon the order of 10 nm). In some embodiments, the heat spreading layercan be from 10 nm to 1000 nm thick. In more specific embodiments, thelayer can be from 20 nm to 500 nm thick. In even more specificembodiments, the layer can be from 50 to 250 nm thick. A dielectricstack can optionally be provided above the conductive layer in order tofurther reduce absorption and thus peak heat load.

In particular, in some embodiments, the operating wavelength, numericalaperture/mode size, materials used for fabrication of the gratingcoupler and specific design of the grating coupler (e.g., binarygrating, blazed grating, focusing grating, etc.) can be varied. Inaddition, the materials and process details for fabrication of the heatspreader can be varied—e.g., any sufficiently thermally conductivematerial that is appropriately process-compatible could be used for theheat spreader. For example, aluminum, tungsten, silicon carbide, copper,indium, tin, titanium nitride, or others can be used depending on theprocess technology. In specific embodiments, the thermally conductivematerial is aluminum. Additional thin film layers can be provided abovethe heat spreader in order to tailor optical performance (e.g.,reflection and absorption for a particular wavelength, polarization,etc.).

The specific dimensions of the heat spreader (e.g., lateral extent andthickness) can be varied to suit relevant design constraints, includingphotonic circuit geometry, materials, and expected power. While areflective layer below a grating coupler can be made of a material thathas relatively high thermal conductivity and thus can itself act as aheat spreader to some extent, the required thickness of such areflective layer from an optical perspective can be quite small (e.g.,10-100 nm); at such thickness, performance as a heat spreader isaccordingly somewhat limited. When the layer thickness is substantiallygreater than required for optical purposes (e.g., 100 nm to 1 μm ormore, depending on the geometry and materials used) heat spreadingperformance can be substantially improved.

Exemplary target waveguide structures are illustrated graphically inFIG. 5, where the structures of FIGS. 5A and 5B do not include heatspreaders, and the structures of FIGS. 5C and 5D include heat spreadersbelow the grating structure. The structures illustrated in FIGS. 5B and5D further include a reflective layer below the coupler to improveefficiency of optical coupling as described above.

Typically the heat spreader will extend from the region below thegrating to the edge of the chip where it is in thermal contact with thecarrier that holds the chip. The contact with the carrier that holds thechip allows for heat on the chip to be transferred off of the chip forthermal management. In some cases the carrier has a heat sink that is inthermal contact with the heat spreader on the chip. In some cases,active cooling is provided to the heat sink. A heat spreader also couldbe used as, or used below, an absorbing interface instead of areflecting interface below the grating coupler. This can beadvantageous, for example, if process tolerances are insufficient toguarantee a desired phase relationship between the incoming beam at thegrating coupler and a reflective layer below the bottom cladding. Wheresuch tolerances are insufficient, coupling efficiency can varyundesirably due to process variation. In this case, higher absorbed heatloads would be expected for a given coupled power, and thus a means ofthermal mitigation becomes even more critical.

For the sake of description, the terms “above” and “below” here refer torelative position of layers for a case in which the input beam isincident from the top of the layer stack, as commonly described. In someembodiments, however, an inverted stack can be used, in which case thebeam is incident from below. In such a case, a heat spreader can stillbe applied to laterally disperse heat and/or aid in its extraction fromthe top of the layer stack.

Example 6 below demonstrates experimentally the benefit of a heatspreading layer in mitigating laser damage at power densities typical ofthose used in the instant devices and systems.

Active Waveguide Coupling

According to another aspect, the instant specification provides opticalsystems comprising an optical source and a target waveguide device,wherein the optical energy from the optical source is actively coupledto the target device. In traditional optical systems containing anoptical source and a target waveguide or fiber optic device, thecomponents are associated using either permanent coupling orconnectorized coupling. For example, in systems where the target opticaldevice is contained within an integrated optical chip, is buriedunderground, or is strung under the ocean in a telecommunications cable,the target device is carefully aligned to the input source or sources(e.g., a laser diode, an LED, or the like) and permanently fixed inplace. This process is expensive, time consuming, and usually involvesglue or other permanent adhesive. The connectorized approach is similarin that it requires the careful alignment of a connector to the targetdevice. In addition, connectorized connections are usually made manuallyby a human operator.

The active coupling approach described herein differs from theconventionally coupled systems in that it involves a target waveguidedevice that is readily inserted and removed from the optical system.There is additionally a premium placed on fast cycle times, with thetarget device being coupled to the optical source as soon as possibleafter its insertion into the system. Although a connectorized approachis clearly more suited for this type of operation than a permanentlycoupled approach, even the connectorized approach typically requireshuman intervention to create the connection. Connectorization also addssignificant cost to the system—in the case of telecommunicationssystems, typically $100 per connector.

An active coupling strategy is usefully applied to any of the coupledsystems described herein. It typically involves a laser path thatincludes motorized beam steering and in some cases also motorized focus,and it also preferably includes a feedback loop. Simple feedback loopsare described in co-owned U.S. Patent Application Nos. 62/133,965 and15/072,146. For example, a waveguide tap fabricated within the targetwaveguide device can be used to split out a small amount of laser powerfrom the guided mode, and the tapped power can be routed to a convenientlocation for collection by a camera or other detector to monitor andadjust the optical coupling through the system. Alternatively, or inaddition, light does not necessarily need to be explicitly coupled outof the device in order to provide feedback. Instead, a camera orientedtoward a specific waveguide region can determine the amount of lightwithin the waveguide, in the same way that waveguide coupling losses areestimated by quantifying the scattering loss along the waveguide.

Another closed-loop feedback alternative for monitoring coupling is tointegrate a detector onto the waveguide itself. Although this approachmay complicate fabrication of the target device and may increase cost(for example, a hybrid flip-chip approach is common but expensive, and amonolithic approach requires wires), such integrated detectors are knownin the art.

For any actively coupled system, the optical source is ideally steerablein x,y, and/or tip/tilt directions, and can additionally be focusable.It can in certain embodiments be advantageous to apply moresophisticated beam shaping to the optical source beam in response to thecoupling efficiency, as measured in the closed feedback loop. Suchactive control over the optical input loosens instrument tolerances onplacement of the target waveguide device within the instrument, ontarget device packaging tolerances and substrate tolerances, and also onwaveguide alignment tolerances (e.g., on mask alignment). Fabricationvariations in waveguide shape and size can also be loosened by anadaptive optical input with a closed-loop feedback. Finally, instrumentdrift tolerances can be significantly loosened with closed-loop adaptiveoptical coupling.

A variety of coupling methods can be used independently for inputting anoptical signal into a target waveguide device. These methods canadditionally or alternatively be used without limitation to coupleoptical signals out of the device, for example to an optical detector,detectors, or the like. The three classic approaches to coupling includetransverse or endfire coupling, prism coupling, and grating coupling.Each of these techniques has certain advantages with respect its use inan optical analytical system. In particular, transverse couplingrequires little or no space on the target device and provides a highlevel of overall coupling efficiency (70-90%). Transverse coupling,however, requires polishing of the side of the target waveguide device,can impact packaging of the device within an optical system, and canrequire sensitive alignment of the target device in three dimensions.Prism coupling also displays relatively high coupling efficiencies(50-80%), but it requires the incorporation of a high-index prism intothe system packaging, space on the surface of the target device, andalignment of the target device with respect to prism tilt. Standardgrating coupling efficiency can be relatively low, but the efficiency issignificantly improved (to 90%) with specific grating profiles andincident beam energy distributions. Grating coupling also requires spaceon the surface of the target device and is sensitive to tilt alignmentbetween the optical source and the target device.

As will be further described in the Examples, the overall couplingefficiency of an optical system is defined as

η=η_(instrument)·η_(target device)·η_(optical source)

where the instrument coupling efficiency (η_(instrument)) describes theratio of power in the guided mode to the total power delivered to thetarget device by the instrument. The denominator includes unused powerthat does not couple into the target device in the form of substratemodes or other:

$\eta_{instrument} = \frac{{Power}\mspace{14mu} {in}\mspace{14mu} {guided}\mspace{14mu} {mode}}{{Total}\mspace{14mu} {incident}\mspace{14mu} {power}}$

where the target device coupling efficiency (η_(target device))describes the ratio of power in the guided mode to the total powercoupled into the device, and where the denominator includes power insubstrate modes which must be prevented from reaching any detectorelements:

$\eta_{{target}\; {device}} = \frac{{Power}\mspace{14mu} {in}\mspace{14mu} {guided}\mspace{14mu} {mode}}{{Total}\mspace{14mu} {power}\mspace{14mu} {in}\mspace{14mu} {device}}$

and where the optical source efficiency describes the fraction of lightcoupled into a guiding layer that can be successfully coupled intoindividual channel waveguides:

$\eta_{opticalsource} = \frac{{Power}\mspace{14mu} {in}\mspace{14mu} {guided}\mspace{14mu} {mode}}{{Total}\mspace{14mu} {power}\mspace{14mu} {planar}\mspace{14mu} {wave}\mspace{14mu} {guide}}$

The values of η_(target device) and η_(optical source) should generallybe considered more important than η_(instrument) within an integratedsystem, because they represent light scattered inside the targetwaveguide device that can increase background signals and thus putpressure on the laser rejection filters and other background mitigationstrategies. Low instrument efficiencies can be compensated for bychanges in instrument design. Exemplary target waveguide design andestimation of coupling efficiency is provided below in Example 2.

Multimode Integrated Coupler

According to another aspect, the instant specification providesmultimode integrated optical coupling devices and optical systemscomprising such devices. As described above, target waveguide devicestypically include a limited number of optical inputs that are coupled toan optical source. Optical energy entering the device is directed bywaveguides to locations of interest within the device through splittersthat are fabricated within “fan-out” regions of the target device. Thedevices disclosed in this section of the disclosure, however, include amultimode coupler element. In these devices, the role of the multimodecoupler element is not to route light to individual output waveguides,but rather to distribute the light into pre-planned “hotspots” wherenanoscale sample wells are located.

The design of the multimode coupler device allows flexibility in thespacing, location, number, and relative brightness of the hotspots.Multimode couplers are a mature technology with a great deal of processdevelopment and design approaches already in place. A photograph of anexemplary 1×8 splitter device is shown in FIG. 6, where hotspots areclearly evident in an arrayed pattern across the surface of the device.A nanowell can accordingly be placed on top of each hotspot.

The design space of such multimode devices is quite flexible, withdevices designed to have different spacing and intensity. In particular,in some embodiments, the devices are designed to display more efficientuse of laser power, lower propagation loss, lower autofluorescence,allow a more flexible layout of nanowells, and use less space on thechip for routing and/or splitting. In some embodiments, a specifiednumber of waveguides are fanned out and illuminated, but the waveguidesare terminated in a multimode coupler structure. In specificembodiments, the structure is square or rectangular, or it could beanother structure that uses space more efficiently, for example withgreater packing density. The use of a multimode coupler could partly orcompletely eliminate the large cascade of splitters necessary in afan-out region to divide a single input waveguide into thousands or moreseparate waveguides for transmitting light to the nanoscale samplewells.

In some embodiments, the multimode couplers are designed to providevarying intensity. For example, the intensity can be programmed tocompensate for scattering loss, propagation loss, loss at the nanowell,and the like.

In some embodiments, the devices are designed to provide programmableexcitation. Such devices are similar to classic waveguide illumination,with optical switches implemented to switch on and off different regionsof the chip. In some embodiments, the devices are designed to providevariable excitation. As is used in classic waveguide illumination,variable optical attenuators (VOAs) can be integrated into differentlines to provide for adjustment of the power density at different groupsof nanowells. Such variable excitation could be used in a “per chip SNR”optimization, where it could be used to adjust power output afterinitial results from subsections of a particular chip. It could also beused to program the chip with a diversity of excitation powers andsimultaneously collect data at different optimization points on thelaser titration curve.

All of the above optical features could be achieved using traditionaloptical trains as well as with classical waveguide illuminationapproaches, but they are far simpler to achieve using a multimodecoupler device. In addition, a multimode coupler overcomes some of theproblems that can arise with traditional optically coupled devices. Forexample, it is generally difficult to space output waveguides as closelytogether as desired because of interference between guided modes.Autofluroescence may also limit the potential SNR of a classicalwaveguide device. The splitters used in a classical waveguide device mayadditionally be problematic in that they require significant amounts ofspace on the device. Traditional splitters may also limit accuracy ofthe device, as each stage adds variability into the different branches.

Polarization Schemes for Efficient Excitation of Nanowells

According to another aspect, the instant specification provides methodsand devices for optimizing the excitation of arrayed nanowells in anoptical analytical device. As described above, analytical reactions,preferably immobilized single template/DNA polymerase sequencingreactions, are excited with laser light, typically near metallicnanostructures. In such systems, the polarization of the optical sourceis an important consideration in implementing the design. In typicalsystems, the input light is linearly polarized due to the properties ofthe optical train. In most circumstances, however, a differentpolarization would be more efficient. Higher efficiency results inbetter uniformity of excitation and lower power requirements forexcitation. Better uniformity improves the quality of data generatedfrom the analytical reaction, and lower power requirements translatesinto lower autofluorescence and lower heat generation.

In the above-described integrated target devices, the nanowells areilluminated by an optical source within the device, typically anexcitation waveguide. The nanowells are preferably cylindrical in shape,wherein the inner walls are commonly formed from a metallic layer, andthe bottom of the nanowell is commonly a glass/water interface. As isknown in the art, the penetration of an evanescent electric field into ametal varies with polarization of the optical source, and there iscorrespondingly a strong polarization dependence for the evanescentfields exciting nanowells in such devices due to the metallic layersurrounding the nanowells. When an enzyme, such as a DNA polymerase, isimmobilized at a specific location within a nanowell, the strength ofthe electric field from the optical source thus varies significantlydepending on the position of the immobilized enzyme, and thus thefluorescent target molecule, within the nanowell.

The instant inventors have discovered that a simple linear polarizationof excitation light generally provides relatively poor field uniformityinside a metallic nanowell, but that the uniformity can be improved byan alternative approach to polarization. In particular, for some systemsusing linearly polarized excitation light, the falloff in excitationenergy can be a factor of two between edge locations aligned with thelaser linear polarization (0° and 180°), and locations orthogonal to thepolarization direction (90° and 270°). For example, as illustrated inFIG. 7, target molecules positioned in a nanowell (i.e., a ZMW) atlocations 1 or 2 experience high laser electric fields when excited bylinear-polarized laser light, whereas those positioned at locations 3 or4 experience significantly lower electric fields. A graph representingthe estimated falloff in electric field along the x and y coordinates isalso shown in FIG. 7. By comparison, circularly polarized light reducesthe variability in the excitation field by half. It should be understoodthat the fluorescence signal varies quadratically with excitationelectric field, so the impact of non-uniformity in excitation field canbe significant.

As an alternative, if a nanowell is excited with circularly polarizedlight, while there is still a falloff between the peak electric fieldlocation in the center of the nanowell compared to the edge, thisfalloff is radial and not as deep. Accordingly, as shown in FIG. 8,target molecules positioned in a nanowell at locations 1, 2, or 3 wouldexperience similar electric fields when excited by circularly-polarizedlight. It should also be noted that other system performance metrics maybe affected in different ways by the target molecule position, and anincreased uniformity of excitation field is but one factor in improvingperformance of the system. However, converting to circularly polarizedlight removes a significant factor that is a function of azimuthal andradial location within the nanowell and thus reduces overall variabilityin the excitation level.

Depending on the particular optical system, the conversion of anexcitation beamlet from linear to circular polarization may be more orless complicated. For a relatively simple case, for example where theexcitation beam is provided in a traditional optical train, theconversion may be effected, for example, by the simple addition of aquarter wave plate at a collimated location in the laser path. Thismodification converts the light at that spot from linear polarization tocircular. The ultimate polarization at the nanowell will be slightlydifferent, however, due to reflections and asymmetric filters. Thedesign of an appropriate waveplate or two that results in true circularpolarization at the nanowell is straightforward, however, as would beunderstood by one of ordinary skill in the art, if the optical designdetails of the lenses and filters in the system are known.

For a more complex and compact optical system, for example where theoptical signal is transmitted through a waveguide, and where a metal isused, as described above, to define the excitation volume and to provideenhancement of the laser field strengths, the field strength may bespatially dependent on the polarization direction. Optical waveguidesare generally polarized, with two possible orientations (TE and TM)which are orthogonal to each other. A slab waveguide can combine TE andTM modes, such that the TE mode can be used to propagate one laserwavelength (532 nm) and the TM mode can be used to propagate a secondwavelength (642 nm). For purposes of the instant disclosure, however, aslab waveguide can be used to create circular polarization, or anapproximation of circular polarization, in the waveguide. This, or aneven more complex polarization scheme, provides maximum uniformity inelectric field across all possible target molecule locations in samplesilluminated by such waveguides. FIG. 9 provides a schematicrepresentation of the effect of target molecule location on excitationby different TE modes.

Furthermore, while waveguides are typically designed for transmission ofeither TM or TE modes, there is a third unique mode definition, TEM,that can be used to transmit optical energy to arrayed nanowells in atarget device. For example, a square embedded guide with the same indexin all cladding directions could simultaneously support both TE and TMtransmission, and if the symmetry is perfect, or nearly perfect, both TEand TM will have identical group velocities. Similarly, a TEM mode canbe used for minimal polarization anisotropy, and hybrid modes in generalcan be constructed quite generally to match a desired polarizationconfiguration. FIG. 10 illustrates how these modes can be combined withdifferent group velocities to create desired electric field patterns ina waveguide.

Multi-Look and Multi-Hotstart Approaches

According to yet another aspect, the instant specification providesdevices and systems for highly arrayed optical analysis in which thetarget nanowells may not necessarily be illuminated simultaneously. Inother aspects, the analytical reaction occurring within the targetnanowells may not necessarily be initiated simultaneously in all of thenanowells.

In some embodiments, the instant integrated target devices involve asingle sequencing experiment per chip, and all nanowells on the deviceare illuminated simultaneously. In other embodiments, however, only halfof the nanowells are illuminated at a time. In still other embodiments,one third, one fourth, or even fewer of the nanowells are illuminated ata time.

In some embodiments, a single “hotstart” initiates polymerase activityin all of the nanowells simultaneously. In other embodiments, polymeraseactivity is initiated at two, three, four, or even more times on a giventarget device. Initiation of polymerase activity may be triggered invarious ways, for example by the addition of an essential component ofthe enzymatic reaction, e.g., one of the four nucleotides in a DNApolymerase-catalyzed reaction, that is initially not present in thesample or that is initially present in limiting amounts. In someembodiments, polymerase activity is triggered by the release of atrapped form of an essential component or by activation of an otherwiseinactive form of the component. In these embodiments, the essentialcomponent could be, for example, one of the four nucleotides requiredfor the DNA polymerase reaction, or could be the DNA polymerase enzymeitself.

The multi-look and multi-hotstart concepts disclosed herein address someof the challenges in the use of integrated waveguide devices for themeasurement of nanoscale analytical reactions. For example,autofluorescence in the waveguide core material, laser scattering lightlevels combined with limited design space for laser blocking filters,heating of the coupling pad due to imperfect coupling efficiency, andlarge laser power required can be problematic. Independent of thewaveguide illumination scheme, the compute bandwidth is an importantengineering problem. The figure of merit for all of these issues isdivided by the number of looks in a multi-look approach (e.g., if a 10 Wlaser is required for single look, 5 W would be required for two-look;if the autofluorescence level is X in a single look, it would be X/2 ina two-look, and so on). Although the use of multi-look approachesdecreases instrument throughput, it can also reduce the cost peranalytical reaction of the device and can also simplify/reduce the costof the instrument.

In terms of waveguide illumination, there are several ways to implementmulti-look excitation. An instrument-centric approach is to includemultiple optical inputs on the target waveguide device, and aim an inputoptical beam at one of these inputs at a time. FIG. 11 illustrates howthis approach could be implemented with two basic coupling schemes.Specifically, FIG. 11A compares the single-look design (top) and a3-look variant (bottom) in a target waveguide device containing gratingcouplers. With the 3-look variant, a single input optical beam is aimedat the three separate input grating couplers in sequence in order toexcite samples along the “Look 1”, “Look 2”, and “Look 3” waveguides,respectively. FIG. 11B shows the corresponding single-look (top) and3-look (bottom) design variants for target devices employing endfirecoupling. The corresponding designs for target devices employing prismcoupling are not shown but would be similar to the designs shown for thegrating-coupler devices of FIG. 11A. Specifically, in the prism-coupleddevices, the input grating couplers of the designs shown in FIG. 11Awould be replaced with input prism couplers.

A further example of the multi-look approach, in particular where theinstrument provides multiple optical beams for illumination of a targetdevice, is illustrated graphically in FIG. 12. A single-look device withinput grating couplers and designed for use with three input opticalbeams is shown in FIG. 12A. A corresponding 3-look device with inputgrating couplers and designed for use with three input optical beams isshown in FIG. 12B. In each case, the three input beams are indicated inthe drawing as ovals positioned to the left of the respective devices.It should be understood, however, that these beams would, in practice,illuminate the input couplers on the devices and be launched into theintegrated optical waveguides in each case. Similar designs could beprepared using prism input couplers in place of the grating inputcouplers. FIG. 12C shows a 3-look endfire-coupled device for use withthree input beams. The input beams in this device are designated by thethree pairs of convergent lines targeting the waveguides. In FIGS. 12Band 12C, movement of the three input beams from look to look isindicated by small arrows.

A variety of on-chip optical switches are also available forimplementing the multilook concept. An efficient and inexpensive exampleis a thermally-activated Mach-Zehnder switch. Since these switches arerelatively slow and display different on/off speeds, they are mostsuitable in instruments where switching times of one or two seconds aresufficient. It should also be noted that on-chip switching isindependent of the coupling scheme. An endfire-coupled target devicewith a single optical input is illustrated in FIG. 13, but correspondinggrating-coupled and/or prism-coupled target devices could likewise bedesigned. As shown in the device of FIG. 13, three Mach-Zehnder switchesare used to control the excitation of four different waveguides toprovide four separate “looks” in this device. A more detailed view of anindividual thermal Mach-Zehnder switch is also shown in FIG. 13. Suchswitches are known in the art and can be readily included in the designand fabrication of an integrated waveguide device.

Polarization can also be used to implement a two-look scheme. The use ofpolarization can advantageously require fewer moving parts or smalleradjustment ranges in the instrument, and less real estate than anon-chip version. An instrument-level implementation of such an approachis depicted in FIG. 14A, and an on-chip implementation is depicted inFIG. 14B. Specifically, the target device shown in FIG. 14A includes apolarization-sensitive beam splitter that is used to route light betweentwo different waveguides (“Look 1” and “Look 2”). The optical input isswitched by the instrument between polarization states (e.g., s and p)for recognition by the beam splitter. The target device shown in FIG.14B includes a polarization-maintaining input waveguide that leads to adegenerate guide. A Pockels cell polarization switch, or the like, isused to modulate the polarization state of light passing through thedevice, and a downstream polarization-sensitive beam splitter routeslight between two different waveguides (“Look 1” and “Look 2”) fortransmission to the respective nanoscale sample wells.

Wavelength tuning can also be used for implementing the multilookconcept. In this approach the laser in the instrument is a tunablelaser, and the optical input is routed through the device according tothe wavelength. A basic arrayed waveguide grating (AWG) device could beused here, with a large number of looks enabled according to establishedAWG technology. An exemplary AWG-implemented target device with sixoutput waveguides is shown in FIG. 15. Note that the excitation sourcecould be tuned, for example in 25 nm increments, with each incrementbeing directed to a different “Look”. Alternatively, a broadband source(e.g., an LED) could be used along with a tunable filter that wouldselect one wavelength at a time. The wavelength step size should bechosen to be small enough that the differences would not have asignificant impact on excitation of the subject analytical reaction.

Fiber Spacing Concentrators and Fiber Alignment

According to yet another aspect, the instant specification providesfiber spacing concentrators with reduced loss and improvedchannel-to-channel uniformity.

Multi-channel microfabricated optical devices are of use intelecommunications applications, for high-speed optical interconnects incomputing, and potentially for bioanalytical applications. Opticalfibers are typically used to transmit signals at the macro scale, andvarious means can be used to couple the signal between a microfabricatedstructure and an optical fiber. However, there is a large mismatchbetween the minimum pitch of coupling structures on a microfabricatedcomponent (which structures can be roughly the size of the optical fibermode and thus spaced on this scale) and the minimum pitch of an array ofoptical fibers (limited by the fiber cladding or coating diameters,which can be 30× the mode diameter or more). From a practicalstandpoint, this means that more area—and thus more cost—must be devotedto coupling structures on the chip than required from an opticalperspective.

A fiber spacing concentrator (FSC) is a planar microfabricated passiveoptical component used to provide well-defined spacing of multipleindividual optical channels with a fixed pitch that can be made muchtighter than the spacing between optical fibers in a fiber array.Embodiments of such FSCs are available commercially. See, e.g., fiberspacing concentrators from Teem Photonics, Meylan, France(http://www.teem-photonics.com/fiber-spacing-concentrator.html). Use ofan FSC for optical coupling allows for much tighter spacing of couplerson the target microfabricated optical device, thus reducing the requiredarea and cost for a given number of channels. However, this benefitcomes at the cost of some loss of optical transmission, which can benon-uniform across the array. Additional power and potentiallyadditional degrees of freedom for power control can be required tocompensate for such non-uniform losses, which ultimately add to systemcost.

From a physical perspective, the FSC consists of three key components: amicrofabricated part in which waveguides are defined, a mechanicalassembly for holding an array of fibers, and the fiber array itself. Thefiber array can be fixed (e.g., bonded) in the mechanical assemblybefore subsequent alignment of the mechanical assembly and bonding tothe waveguide component.

A large fraction of the losses in an FSC assembly likely arise from thespatial mismatch between the waveguide structures in the microfabricatedcomponent and the locations of the cores of the individual fibers in thearray. While the main component of the FSC is lithographically patternedto nanometer-scale accuracy, the array of spots from the fiber array ismechanically defined. Errors in spot position can arise frommanufacturing tolerances in the array of V-grooves used to hold thefibers, which can be sub-micron for a part also made lithographically,as well as from core-cladding concentricity errors of the individualfibers, which can be substantial on the scale of the spot diameter (e.g.1 μm concentricity error with 3.4 μm mode field diameter for asingle-mode fiber in a visible wavelength). Exemplary V-grooveassemblies, and their alternatives, are described in U.S. Pat. No.7,058,275.

To reduce the loss of optical throughput in the FSC, as well as toimprove uniformity among channels in the FSC, it would be advantageousto better control the spacing of the fiber modes at the interfacebetween the fiber array and the waveguide structure. This might be donewith active control of individual fiber position at assembly, but thechallenges of simultaneously fixturing many small fibers for activealignment and subsequent bonding in place in a very restricted volume(with fiber spacing on the order of the fiber diameter) are difficult.

To improve uniformity and reduce losses in an FSC assembly, the barefibers in the mechanical assembly (for example in a V-groove array) canbe replaced with pre-aligned fiber and ferrule assemblies that can offermuch tighter concentricity. See FIG. 16. Active alignment of individualfibers to ferrules is an existing process capable of providing very lowloss in fiber-to-fiber links. Concentricity tolerances can be reducedfrom ˜1 μm for bare fiber to ˜125 nm between the core and aprecision-polished ferrule. Suitable ferrules and core alignmenttechnologies are available commercially, for example, from Diamond SA,Losone, Switzerland. This approach substantially reduces the overallalignment error between fiber core and waveguides in the FSC, resultingin improved uniformity and lower transmission losses.

Various aspects of the devices can be varied including:

-   -   The operating wavelength, fiber mode field diameter, and type.    -   Number of inputs to the FSC—this approach is readily applicable        to an FSC with arbitrary channel count.    -   Details of the active alignment technique for individual inputs        in the FSC. Commercial products are available with a pre-aligned        ferrules that are readily incorporated into an integrated        solution with only minor changes to V-groove geometry/spacing.    -   Design of the microfabricated portion of the FSC.    -   Removal of the microfabricated portion of the FSC, leaving the        V-groove array with pre-aligned fibers. This alternative        provides an accurately spaced array of spots on a large pitch        for any application where it is appropriate.    -   Materials of the V-groove array (glass or silicon or otherwise),        and methods of assembly (e.g., adhesive bonding or mechanical        fastening).

Fiber spacing concentrators are available commercially, where losses areon the order of 1 dB for applications in typical telecom wavelengths inthe near IR. Losses would increase for visible wavelengths usingexisting devices, as the sensitivity to a given degree of mechanicalmisalignment increases with decreasing spot size/MFD. The approachesdescribed here improve the throughput losses and non-uniformity ofexisting FSCs.

According to yet another aspect, the instant specification furtherprovides innovative approaches to the alignment and connection ofoptical fibers. In particular, these approaches relate to the use of anactive actuator to complete the interconnection. Such approaches can below cost and easy to use.

As is known, low power and low power density fiber modes can beeffectively coupled through precision ferrules and passive matingsleeves. High power, high power density, and small mode field diameterfibers are more challenging for passive interconnection, however, owingboth to risk of damage from contamination and tight tolerances.

Passive free space interconnects have been used in order to couple withlow risk of damage. These interconnects are, however, typicallyexpensive and time consuming to use. Passive physical contactinterconnections are well known for telecommunication applications. Thephysical contact interconnects are not well suited for high powervisible light applications, for which even minute contamination can leadto a runaway that causes destruction of the fiber (aka fiber fuse), ormay result in less catastrophic but still substantial reductions intransmission.

High power fibers use end caps, a fused unguided section to expand themode and increase the threshold against damage from contamination.Unfortunately, this end cap also precludes the use of efficient physicalcontact connectors for the same reason.

Free space interconnections for fibers with end caps are availablecommercially. Such devices can be based, for example, on mechanicalactuation driven by manual lead screws. The aligned optic can be, forexample, a pair of mirrors. While such approaches can be effective, theyare expensive and require skilled labor time to align at each fiberinsertion.

An alternative is the use of an active optical element to match theexpanded modes between two such single mode devices. This can make useof optics to create multiple beams to guide the alignment (e.g.,diffractive optical elements (DOEs)) or other servo features. A devicesuch as the Varioptic Baltic 617 or similar can be effective in matchingmodes to ensure an efficient, low cost interconnect with good toleranceto contamination.

The active optical device can be based on different actuation methods(EAP, VCM, PZTs, etc.). The device can be based on scanning prisms(e.g., Risley pair), though these may be more costly. Methods based ondiffraction gratings, real time or not, can also be used.

Integrated System-On-Chip

In another aspect, the instant specification provides waveguide devicesthat include an integrated optical source, where the optical source iseither fabricated within the waveguide device itself or is attached tothe device after fabrication. The previously described opticalanalytical systems typically comprise an optical source (or sources)(e.g., a PLC) that is physically separate from the target waveguidedevice. Optical energy emitted from the source is therefore coupled tothe target device through free space, as described in detail above. Insome circumstances, however, it may be advantageous for the opticalsource to be integrated into the target device package, for exampleusing a multichip module or system in a package (SIP) approach. Suchapproaches are well known in the electronics industry but have notpreviously been applied to integrated waveguide devices such as thoseused in multiplexed DNA sequencing chips. By integrating a laser, orother suitable optical source, directly into the chip package, each cellbecomes a self-contained optical bench capable of illuminating andviewing target molecules within an array of optically coupled nanowells.

Conventional SIP approaches can accordingly be adapted for use in theinstant integrated systems, for example by modifying a waveguide deviceusing flip-chip assembly techniques, or the like, for example to mount alaser diode chip or other compact optical source directly on thewaveguide device. Flip-chip bonding techniques have been usedextensively in the electronics industry, including their more recentapplication to optoelectronics components. See, e.g., Han et al. (1998)J. Electron. Mater. 27:985; Li et al. (2004) P. Elecr. C. 2:1925.Advantageously, flip-chip techniques can make use of solder bumps formounting components on interconnects. Solder bumps may, upon reflowing,pull the components into position due to the surface tension of themolten solder, thus facilitating the alignment of optical componentsduring assembly. The choice of optical source will depend on the needsof the system. Although traditional laser diodes are edge emitters andmay therefore require more complex assembly arrangements, newertechnologies, such as, for example, vertical cavity surface emittinglaser (VCSEL) technologies, enable more direct optical coupling from thesource to the waveguide device.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the devicesand systems described herein can be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following Examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1. Binary Grating Couplers with Low Numerical Aperture

This example describes the design, optimization, and modeling of variousbinary grating couplers having low NA. The coupling of optical energythrough free space to a 2-dimensional grating coupler can be modeledusing finite-difference time-domain (FDTD) numerical analysis of theMaxwell equations, for example using computer software from Lumerical(www.lumerical.com) or the like. An example of such modeling is shown inFIG. 17A, where the 2-dimensional Gaussian light source (1702) is shownin light shading above the device model. The arrow shown within thelight source represents a coupling angle of 10 degrees. The arrow isshown intersecting a rectangular box that represents the grating couplerstructure (1704). The oxide cladding (1706) is the solid layersurrounding the coupler. The waveguide core (1708) is represented as athin line extending to the left from the coupler. Optical energy iscoupled from above the structure through the grating coupler into thewaveguide core. FIG. 17B shows the results of the FDTD simulation,showing the light (in power units) coupling through the grating andpropagating to the left down the waveguide core.

FIG. 18 summarizes the structural features of various binary gratingcoupler designs and compares the FDTD-modeled coupling efficiencies forthose designs. The designs correspond to those described in FIGS. 3C-F.FIG. 19 shows the results of FDTD modeling of grating couplers havingstructures corresponding to that of FIG. 3A with different numericalapertures (NA). Beam sizes and grating sizes were varied in the modelsto be consistent with the numerical apertures.

FIG. 20 illustrates the impact of numerical aperture on the alignmenttolerances for beam and grating pairs. As is clear from the models, theefficiency of coupling for the low NA couplers is much less sensitive toalignment between the optical source and the grating coupler compared tocoupling for the high NA couplers.

FIG. 21 compares the modeled effects of grating period (A), buried oxidethickness (B), duty cycle (C), and etch depth (D) on efficiency ofcoupling. As shown in FIG. 21A, the coupling efficiency is sensitive tochanges in the grating coupler period, and when the numerical apertureis decreased, the coupling efficiency becomes even more sensitive tovariations in the period. Since the period is mainly determined by theaccuracy of lithography and masking during chip fabrication, however,these variations can be well controlled. It should also be noted thatthe sensitivity of coupling efficiency on period also shows angulartolerance. Smaller numerical apertures correspond to tighter angulartolerance.

FIG. 21B demonstrates that coupling efficiency is very dependent on thethickness of the bottom oxide cladding. This dependence on bottom oxidecladding thickness is observed at all values of numerical aperture.Without intending to be bound by theory, it is believed that thisdependence results from reflection of optical energy from the siliconsubstrate.

FIG. 21C shows that coupling efficiency is relatively insensitive tochanges of grating coupler duty cycle for couplers with high numericalaperture, but the coupling efficiency becomes more sensitive to changesin duty cycle as the numerical aperture is decreased. Likewise, as shownin FIG. 21D, coupling efficiency is relatively insensitive to changes ofgrating coupler etch depth at high numerical aperture, but thesensitivity to etch depth variation increases for lower numericalapertures.

FIG. 22 summarizes simulations for couplers designed using parametersobtained from the simulations of FIG. 19. The bottom three rows showresults using these parameters in simulations using an etch depth of 115nm. The optimal bottom oxide thicknesses are as shown in the bottom rowof the figure.

Example 2. Estimation of Coupling Efficiencies into Model TargetWaveguide Device

This example provides estimated coupling efficiencies for a waveguidedevice with an Si₃N₄ core with dimensions roughly 0.600×0.050 μm,surrounded by SiO₂ cladding, and supporting a single TM₀ mode.

-   Coupling efficiency:

$\eta_{{target}\; {device}} = \frac{{Power}\mspace{14mu} {in}\mspace{14mu} {guided}\mspace{14mu} {mode}}{{Total}\mspace{14mu} {power}\mspace{14mu} {in}\mspace{14mu} {device}}$

-   Waveguide effective index:

$n_{e} = {\frac{\beta}{k} = {1.9}}$

-   λ=532 nm, k=1.18×10⁻⁹ cm⁻¹-   β=1.87×10⁵ cm⁻¹-   κ=coupling coefficient (mode overlap integral)-   Maximum condition: κL=π/2-   Requirement for minimum radius of curvature: 0.9 mm-   Radiative loss per bend: 0.5 dB

A not-to-scale representation of the exemplary waveguide cross-sectionis shown in FIG. 23A. Estimates of coupling efficiency are based on acalculation of the overlap integral between the desired mode profile andthe excitation field. An analytics solution of the fields for thisgeometry is not known, but the basic mode profile of the TE₀ mode ofthis waveguide can be approximated (Schlosser and Unger, based onassumption of large aspect ratio). The electric field intensity throughthe center of the waveguide is plotted in FIG. 23B (Schlosserapproximation).

Example 3. Theoretical Transverse Coupling into Waveguide Device with aPolished Facet

The overall coupling efficiency of a device with a polished facet is theproduct of reflectance loss and mode overlap, where reflection loss forfree-space coupling is larger than for an incident plane wave: 9.6%.Perfect coupling would require an incident energy distribution that isexactly the inverse of the far-field distribution of light exiting theguide. A more accurate calculation of the reflectance loss, however,would require integration over these angles. The result of integratingover the high NA dimension only is 12.4%. The best-case insertion lossof the device under a straightforward approach is

η_(instrument)=0.876

The efficiency could be improved by applying an AR coating. Theefficiency could also be improved by including a very small air gap—onthe order of the light wavelength—between the target device and the exitfacet of the illumination source.

Efficiencies are determined by the mode overlap integral:

$\eta = \frac{\left\lbrack {\int{\int{{A\left( {x,y} \right)}{B^{*}\left( {x,y} \right)}dxdy}}} \right\rbrack^{2}}{\int{{A\left( {x,y} \right)}{A^{*}\left( {x,y} \right)}dx{\int{{B\left( {x,y} \right)}{B^{*}\left( {x,y} \right)}dxdy}}}}$

Simulations for prototype coupled waveguide devices are shown in FIG.24, where the left panel shows a mode profile for a simple channelguide, and the right panel shows the same channel guide with an addednanohole is added. A small perturbation to the field profile isnoticeable at the center top edge, but this perturbation was ignored forcoupling estimates.

In principle, an input optical beam can be created with very good matchto the mode profile. As a limiting case, it can be assumed that theoverlap integral is perfect for perfect alignment. In this case thesensitivity to alignment can be estimated by a calculation of theoverlap integral as a function of beam displacement. Since the degree ofconfinement in the y direction is much stronger than in x, only ymisalignment can be considered. Specifically, the spatial scale of ymisalignment impact is roughly 5× larger than for x misalignment, and itis easier to mitigate with in-plane tapering of the guide input section.

The impact of y misalignment is calculated from the mode overlapintegral and illustrated in FIG. 25. At 100 nm misalignment, the powerdrops by roughly half. If high device efficiency is needed, or if a lowdrift in intensity at measurement locations on the target device isneeded, active alignment may be necessary. It may also be worthconsidering increasing the beam size in order to loosen the mechanicalrequirements for achieving a certain minimal field intensity at themeasurement locations on the target device, but an increased beam sizewill not change the ratio below, nor will it change the tolerance on agiven power stability requirement. A flattop intensity profile could beconsidered; in such a configuration a gradual drop in intensity isavoided at the expense of a rapid falloff at the edge of a “safe” range.

Example 4. Theoretical Coupling into Waveguide Device Using a PrismCoupler

An optical waveguide confines light in the x and y dimensions; theconfinement requires total internal reflection and a cladding with lowerindex than the core. Coupling into a target waveguide device by simplerefraction is not possible. The geometry of coupling is constrained byphase-matching between the free-space optical source beam and the guidedmode according to:

$\beta_{m} = {\frac{2\pi n_{p}}{\lambda}\sin \theta_{m}}$

Assuming a perfectly collimated input beam with diameter W, θ_(m) is theincident angle of the input beam inside the prism. The couplingcoefficient, κ, is determined by mode overlap similar to the descriptionin Example 2. The coupling efficiency, η, is determined by κ and theinteraction length, L. Finally, weakly coupled modes are assumed.

It is theoretically possible to achieve 100% coupling efficiency in thisarrangement with a perfectly controlled air gap and waveguide tolerancesand with a flattop incident beam. In practice, however, couplingefficiencies of 90% have been demonstrated in the laboratory. Suchefficiencies have required a non-Gaussian beam and a tapered air gap. Ina straightforward approach with a uniform air gap and a Gaussian beam,efficiencies very close to the 81% theoretical limit have beendemonstrated. The tolerances required for this approach in this exampleare as follows:

-   Air gap: 30 nm-   Air gap variation=0-   z alignment accuracy: 50 nm-   y alignment accuracy: 50 nm*cos θ_(m)-   For perfect geometry complete coupling occurs at an interaction    length,

$L = {\frac{w}{\cos \theta_{m}} = \frac{\pi}{2\kappa}}$

It should be understood that misalignment in the z direction willprevent complete coupling. Furthermore, complete coupling can only occurfor a flattop beam, whereas a Gaussian beam is theoretically limited to80% efficiency, even for a perfect geometry. If the efficiencyrequirement is relaxed to 60%, the tolerances become much looser.Accordingly, for the instant example,

η_(instrument)·η_(device)=0.6, with η_(device)=0.080.

It has been noted that the prism must have a higher refractive indexthan the cladding material. This requirement is very general, butmaximum coupling efficiencies and instrument configurations aredependent on the prism index selected. A higher index implies a lowerincident angle, which is convenient for flexibility in instrument anddevice packaging, and higher theoretical coupling efficiencies. Forexample, FIG. 26 illustrates the relationship between prism refractiveindex and the input incident angle for a prism-coupled device, where theeffective refractive index of the device is 1.58.

Example 5. Theoretical Coupling into Waveguide Device Using a GratingCoupler

The efficiency of a grating-coupled target device is fundamentally lowerthan for a transverse-coupled or prism-coupled device—typically 10% fora simple grating structure. Significantly, a grating coupler lacks thechief advantage of prism coupling, which allows the incident energy tobe largely confined to a single mode. In particular, zero order energypasses directly into the substrate with a grating coupler, as do many ofthe nonzero orders. Additionally, no total internal reflectance meansstrong coupling, each waveguide mode has a complete set of spatialharmonics underneath the grating, and the grating itself has higherorders. The efficiency of a grating coupler can be improved byfabricating complicated grating profiles. For example, high efficiencycan be put into one order to improve the coupling. Furthermore, the zand y mechanical tolerances are very similar to the prism coupling case,with the difference being that light is more quickly coupled intosubstrate modes in the grating case as the beam is misaligned.

The basic phase-matching condition for a grating of period d is

$\beta = {{\frac{2\pi}{\lambda}\cos \theta} - \frac{2\pi}{d}}$

Phase-matching can be achieved over a wide range of angles and gratingperiods, so strictly speaking there is flexibility in choice of gratingperiod. Instrument considerations argue for larger incident angles,however, whereas target device space considerations argue for smallerincident angles. FIG. 27 illustrates the relationship between thegrating period and the input incident angle for a device of thisexample, where the effective refractive index of the device is 1.58.

TABLE 2 Summary of the best-case coupling parameters for three exemplarycoupling approaches. Transverse Prism Grating η_(device) 1.0 0.80η_(optical source) 0.68 0.68 0.68 η_(instrument) 0.88 0.96 ymisalignment (3 dB) 110 nm X misalignment (3 dB) 670 nm

Example 6. Laser-Induced Damage due to Heating on a Target WaveguideDevice

As described above, target waveguide devices may be susceptible tothermal damage due to the high intensities of excitation energy neededto illuminate the large numbers of nanoscale reactions being analyzed ina high-density waveguide array. This example demonstrates the protectiveeffect of including a heat spreading layer within the target device.

FIG. 28 illustrates the test setup and shows the power densities oflasers with various numerical apertures. As is apparent in this figure,even with a low numerical aperture (e.g, 0.01) and large beam size(e.g., 33.87 μm), a 100 mW laser will still have a relatively high powerdensity (e.g., 1.11×10⁴ W/cm²). The power densities used in the testsetup were therefore chosen to simulate this range (e.g., 5 to 780 mWlaser power; corresponding to 1.38×10² to 2.15×10⁴ W/cm²). The figurealso illustrates from below and in cross-section the sample used inthese tests. Specifically, the Si substrate was coated with a 2 μm layerof SiO₂, a 0.5 μm layer of amorphous Si, another 2 μm layer of SiO₂, andfinally a 100 nm layer of Al. The sample also included 8 windows etchedthrough the Si layer. For reference, the thermal conductivities of SiO₂,Si, and Al are 1.4, 149, and 240, respectively.

Both surfaces of the samples were visually inspected under a microscopeprior to illumination with various intensities of laser energy. In thefirst experiment, the laser was directed through the window in the Silayer to target the SiO₂ layer, as indicated by the arrow the structuraldiagram of FIG. 29A. Illuminating the sample for 5 minutes at either 5mW of power or 50 mW of power caused no damage, but the sample wasinstantly damaged upon illumination with 100 mW of laser power. The SiO₂sides of the three samples are shown in the top row of FIG. 29B, and thedamage to the A1 side of the 100 mW sample is shown in the bottom row ofthe figure. In the second experiment, the laser was directed to the A1side of the sample in the region of the etched window, as indicated bythe arrow in the structural diagram of FIG. 29C. In this experiment, a 5minute illumination at 100 mW laser power caused no damage, whereasdamage was observed instantly at 500 mW laser power. These samples areshown in FIG. 29D. A third experiment was similar to the second, wherethe laser was directed to the A1 side of the sample in the region of anetched window, as indicated by the arrow in the structural diagram ofFIG. 29E. Laser outputs of 200 mW, 300 mW, and 400 mW were applied tothe sample with no visible damage. Illumination of the same with 450 mWof laser power, however, resulted in damage within 3 seconds. Thissample is shown in FIG. 29F. A final experiment was run, where the A1side of the sample was illuminated by the laser in a region at adistance from a window through the Si substrate, as indicated by thearrow in the structural diagram of FIG. 29G. In this experiment, nodamage was observed at either 500 mW or 780 mW laser power.

Example 7. Simulation of Optimal Waveguide Dimensions for Single-ModeOperation

FIG. 30 shows a simulation of waveguide dimensions meeting single-modeconditions for two different core materials (SiN, top; TiO, bottom) at552 nm. The upper left and lower right insets show FDTD simulationresults for a thin and wide waveguide and a thick and narrow waveguide,respectively. The Lumerical 2D simulation setup is illustrated in FIG.32A, and the power coupling simulation results are shown in FIG. 32B.

Example 8. Simulation of Grating Coupler Designs with Titanium OxideCore

A grating coupler with a titanium oxide core and high numerical aperture(NA=0.13) but otherwise similar in design to the grating couplerdescribed in Example 1 and modeled in FIG. 17A has been simulated byFDTD numerical analysis at two wavelengths. The input beam (at either532 nm or 552 nm) has a beam waist of 1.75 μm (beam MFD=3.5 μm), asource size of 7 μm, and a fiber coupling angle of 10 degrees (with noangle tuning during the optimizations). The Gaussian profile for theinput beam is illustrated in FIG. 31. Geometrical, mechanical, andoptical specifications for a corresponding single-mode fiber (460HP) areavailable, for example, from Thorlabs, Inc., Newton, N.J., USA(www.thorlabs.us). The setup for the FDTD 2D simulation using Lumericalsoftware is shown in FIG. 32A, and the simulated power coupling resultsare shown in FIG. 32B.

Modeling of the coupling efficiency for a high NA grating coupler centerdesign with a titanium dioxide waveguide core at various wavelengths ofinput light is shown in FIG. 33. In this simulation, the coupler wasmodeled using the parameters listed in the second column of Table 3.

TABLE 3 High NA grating coupler center design features for simulationsat 552 nm and 532 nm. Parameters for 552 nm Parameters for 532 nmsimulations simulations Waveguide core TiO₂ (n = 2.55) TiO₂ (n = 2.55)Waveguide cladding SiO₂ (n = 1.46) SiO₂ (n = 1.46) Waveguide thickness100 nm 100 nm Grating coupler number of periods 20 20 Al reflectorthickness 100 nm 100 nm Top cladding thickness 220 nm 200 nm Gratingcoupler period 315 nm 300 nm Grating coupler teeth width 157 nm (dutycycle = 50%) 150 nm (duty cycle = 50%) Grating coupler etch depth 55 nm55 nm Reflector distance 320 nm 290 nm Optimal coupling efficiency 78.4%(−1.06 dB) 78% (−1.08 dB) Fiber x position 1.6 μm 1.8 μm Fiber yposition 1.2 μm 2 μm

FIG. 34A illustrates the relationship between coupling efficiency andthe grating coupler period at an input wavelength of 552 nm, and FIG.34B illustrates changes in coupling efficiency as a function of gratingcoupler period and input wavelength for the simulated design.

FIG. 35A illustrates the relationship between coupling efficiency andthe grating coupler duty cycle at an input wavelength of 552 nm, andFIG. 35B illustrates changes in coupling efficiency as a function ofgrating coupler duty cycle and input wavelength for the simulateddesign.

FIG. 36A illustrates the relationship between coupling efficiency andthe grating coupler etch depth at an input wavelength of 552 nm, andFIG. 36B illustrates changes in coupling efficiency as a function ofgrating coupler etch depth and input wavelength for the simulateddesign.

FIG. 37A illustrates the relationship between coupling efficiency andthe reflector distance at an input wavelength of 552 nm, and FIG. 37Billustrates changes in coupling efficiency as a function of reflectordistance and input wavelength for the simulated design.

FIG. 38A illustrates the relationship between coupling efficiency andthe top cladding thickness at an input wavelength of 552 nm, and FIG.38B illustrates changes in coupling efficiency as a function of topcladding thickness and input wavelength for the simulated design.

FIG. 39A illustrates the relationship between coupling efficiency andthe waveguide core refractive index at an input wavelength of 552 nm,and FIG. 39B illustrates changes in coupling efficiency as a function ofwaveguide core refractive index and input wavelength for the simulateddesign.

Modeling of the coupling efficiency for a high NA grating coupler centerdesign with a titanium dioxide waveguide core using 532 nm input lightis shown in FIG. 40. In this simulation, the coupler was modeled usingthe parameters listed in the third column of Table 3.

FIG. 41A illustrates the relationship between coupling efficiency andthe grating coupler period at an input wavelength of 532 nm, and FIG.41B illustrates changes in coupling efficiency as a function of gratingcoupler period and input wavelength for the simulated design.

FIG. 42A illustrates the relationship between coupling efficiency andthe grating coupler duty cycle at an input wavelength of 532 nm, andFIG. 42B illustrates changes in coupling efficiency as a function ofgrating coupler duty cycle and input wavelength for the simulateddesign.

FIG. 43A illustrates the relationship between coupling efficiency andthe grating coupler etch depth at an input wavelength of 532 nm, andFIG. 43B illustrates changes in coupling efficiency as a function ofgrating coupler etch depth and input wavelength for the simulateddesign.

FIG. 44A illustrates the relationship between coupling efficiency andthe reflector distance at an input wavelength of 532 nm, and FIG. 44Billustrates changes in coupling efficiency as a function of reflectordistance and input wavelength for the simulated design.

FIG. 45A illustrates the relationship between coupling efficiency andthe top cladding thickness at an input wavelength of 532 nm, and FIG.45B illustrates changes in coupling efficiency as a function of topcladding thickness and input wavelength for the simulated design.

The above simulations demonstrate that grating couplers having waveguidecores with relatively higher refractive indices (e.g., n_(core)≥ about1.9) are suitable for the efficient coupling of an input light beam intoa target waveguide device at wavelengths above 532 nm. In particular,the design features of the grating couplers in such target devices canbe modulated in in order to maximize coupling efficiencies of opticalbeams with wavelengths where fluorescent DNA sequencing reagents havemaximal absorbance (e.g., about 552 nm). The simulations can also beperformed using input beams and input grating couplers having lower NAvalues, as would be understood by those of ordinary skill in the art.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

1-95. (canceled)
 96. An integrated optical delivery device comprising:an optical input; a plurality of optical outputs; and a plurality ofoptical output waveguides disposed on a substrate and opticallyconnected to the optical input and the plurality of optical outputs;wherein at least one optical output waveguide comprises a powermodulator.
 97. The integrated optical delivery device of claim 96,wherein the device comprises a plurality of optical inputs, and whereinthe plurality of optical output waveguides are optically connected tothe plurality of optical inputs and the plurality of optical outputs.98. The integrated optical delivery device of claim 96, wherein thepower modulator is a variable optical attenuator.
 99. The integratedoptical delivery device of claim 98, wherein the variable opticalattenuator is an integrated optical Mach-Zehnder interferometer. 100.The integrated optical delivery device of claim 96, wherein the opticaloutput is configured for optical coupling to a target waveguide devicethrough free space at a distance of at least 1 mm, at least 2 mm, atleast 3 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 30mm, at least 50 mm, or at least 100 mm.
 101. The integrated opticaldelivery device of claim 96, wherein the optical output has a numericalaperture of no more than 0.1, no more than 0.08, no more than 0.05, nomore than 0.03, no more than 0.02, or no more than 0.01.
 102. Theintegrated optical delivery device of claim 96, wherein the device isconfigured to illuminate a footprint on a target waveguide device with apower of at least 1 mW, at least 2 mW, at least 3 mW, at least 5 mW, atleast 10 mW, at least 20 mW, at least 30 mW, at least 50 mW, or at least100 mW.
 103. The integrated optical delivery device of claim 96, whereinthe device further comprises a splitting element optically coupled tothe optical input through an optical input waveguide and to theplurality of optical outputs through the plurality of optical outputwaveguides.
 104. The integrated optical delivery device of claim 103,wherein the device comprises at least two, at least four, at least six,or at least eight optical outputs and optical output waveguides. 105.The integrated optical delivery device of claim 103, comprising aplurality of splitting elements, each splitting element opticallycoupled to one of a plurality of optical inputs through one of aplurality of optical input waveguides and to the plurality of opticaloutputs through the plurality of optical output waveguides.
 106. Theintegrated optical delivery device of claim 96, wherein at least oneoptical output waveguide is a tap waveguide.
 107. The integrated opticaldelivery device of claim 106, wherein the device further comprises apower monitor optically coupled to the tap waveguide.
 108. Theintegrated optical delivery device of claim 107, wherein the powermonitor controls a power output transmitted by at least one opticaloutput.
 109. The integrated optical delivery device of claim 96, whereinat least one optical output waveguide comprises a phase modulator. 110.The integrated optical delivery device of claim 96, wherein at least oneoptical output waveguide comprises a mode stripper.
 111. The integratedoptical delivery device of claim 96, wherein at least one optical outputwaveguide comprises a frequency modulator.
 112. The integrated opticaldelivery device of claim 96, wherein the device is configured foroptical output of visible light to a target waveguide device.
 113. Theintegrated optical delivery device of claim 112, wherein the visiblelight has a wavelength in the range from about 500 nm to about 600 nm.114. An analytical system comprising: the integrated optical deliverydevice of claim 96; and an integrated target waveguide device; whereinthe integrated optical delivery device is optically coupled to thetarget waveguide device through free space.
 115. The analytical systemof claim 114, wherein the system further comprises an external opticalsource.
 116. The analytical system of claim 115, wherein the externaloptical source is a laser source.
 117. The analytical system of claim114, wherein the integrated target waveguide device is a multiplexed DNAsequencing device.
 118. The analytical system of claim 117, wherein themultiplexed DNA sequencing device is configured to sequence DNA in atleast 100 reaction regions.
 119. The analytical system of claim 114,wherein the target waveguide device is removable.
 120. The analyticalsystem of claim 114, wherein the system comprises a plurality of targetwaveguide devices.
 121. The analytical system of claim 114, wherein theintegrated optical delivery device is optically coupled to the targetwaveguide device through free space at a distance of at least 1 mm. 122.The analytical system of claim 114, wherein the integrated opticaldelivery device emits at least four light beams.